research paper on protein electrophoresis

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings

Preview improvements coming to the PMC website in October 2024. Learn More or Try it out now .

• Advanced Search
• Journal List
• Polymers (Basel)

On the Applicability of Electrophoresis for Protein Quantification

Karina dome.

1 Institute of Solid State Chemistry and Mechanochemistry, Siberian Branch, Russian Academy of Sciences, 630128 Novosibirsk, Russia; ur.liam@65aiozka (Z.A.); [email protected] (A.B.); ur.csn.dilos@vomol (O.L.)

Zoya Akimenko

Aleksey bychkov.

2 Department of Business, Novosibirsk State Technical University, 630073 Novosibirsk, Russia

Yuri Kalambet

3 Ampersand Ltd., 123182 Moscow, Russia; ur.dnasrepma@tebmalak

Oleg Lomovsky

Associated data.

The data presented in this study are available on request from the corresponding author.

Polyacrylamide gel electrophoresis is widely used for studying proteins and protein-containing objects. However, it is employed most frequently as a qualitative method rather than a quantitative one. This paper shows the feasibility of routine digital image acquisition and mathematical processing of electropherograms for protein quantification when using vertical gel electrophoresis and Chrom & Spec software. Both the well-studied model protein molecules (bovine serum albumin) and more complex real-world protein-based products (casein-containing isolate for sports nutrition), which were subjected to mechanical activation in a planetary ball mill to obtain samples characterized by different protein denaturation degrees, were used as study objects. Protein quantification in the mechanically activated samples was carried out. The degree of destruction of individual protein was shown to be higher compared to that of the protein-containing mixture after mechanical treatment for an identical amount of time. The methodological approach used in this study can serve as guidance for other researchers who would like to use electrophoresis for protein quantification both in individual form and in protein mixtures. The findings prove that photographic imaging of gels followed by mathematical data processing can be applied for analyzing the electrophoretic data as an affordable, convenient and quick tool.

1. Introduction

Protein chemistry methods are currently used to control, optimize, and elaborate novel technologies in molecular biology, pharmacology, bioengineering, and food technology [ 1 , 2 , 3 , 4 , 5 ]. Such efficient, fast, illustrative, and reproducible methods as high-performance liquid chromatography (HPLC) with different detectors and polymerase chain reaction (PCR) coupled with Sanger sequencing are used for protein quantification [ 6 , 7 , 8 , 9 ]. Despite the rapid progress in fast and efficient techniques employed for protein identification and quantification, simpler and more accessible analytical techniques (e.g., the conventional colorimetric measurements) also remain relevant [ 4 , 10 , 11 ]. Thus, these methods are used for Lowry protein assay in solutions in a reaction with the Folin reagent [ 11 ] or Bradford protein assay with Coomassie dye [ 12 ].

Protein-containing objects are usually analyzed by 1D and 2D polyacrylamide gel electrophoresis (PAGE), with sodium dodecyl sulfate (SDS) used as a detergent [ 10 , 13 , 14 , 15 , 16 , 17 ]. Staining with dyes that bind irreversibly to protein molecules but do not form stable bonds with polyacrylamide gel is often employed for protein detection in the gel [ 18 , 19 , 20 ]. The intensity of stained bands in gel depends on the amount of the applied sample; that is, it is assessed according to the laws of colorimetric measurements: staining intensity is directly proportional to protein content.

Electropherograms are illustrative and informative. However, this technique is most typically used as a qualitative method and quite rarely as a semi-quantitative test (only a visual assessment of band staining intensity is performed). The colorimetric approach (usually the visual one) is also employed in individual cases typically related to molecular biology for measuring the resolution during protein separation in polyacrylamide gel, as well as for protein quantification. In the quantification assay, electrophoretic separation is used together with enzyme-linked immunosorbent assay or western blotting [ 21 , 22 ], which requires respective immune sera against the target proteins.

In recent practical studies, there is demand for protein quantification in complex systems containing numerous impurities of protein and non-protein nature. Previously, polyacrylamide gel electrophoresis was used to determine the depth of hydrolysis of pea seed proteins [ 23 ]. The resulting hydrolysate enriched with free amino acids and peptides was used as a component of functional foods. The method combines the qualitative and quantitative assays of a protein mixture by polyacrylamide gel electrophoresis and simultaneous assessment of concentrations of the mixture components. It can also be used to develop special nutrition products containing pea seed proteins [ 24 ]. The topic of creating food products from peas is well developed, products containing peas have been mastered by the food industry and are popular [ 25 , 26 ]. In particular, this review notes the positive aspects of the technologies of dry processing of pea seeds.

Electrophoresis in polyacrylamide gel is no less popular in pharmaceuticals. Thus, in order to optimize the procedure for analyzing the drug aprotinin, the time-consuming chemical analysis was replaced by an analysis using HPLC [ 27 ]. Meanwhile, as aprotinin derivatives have a protein nature, they can be analyzed by polyacrylamide gel electrophoresis. The target aprotinin and its impurities can be detected by gel electrophoresis as clearly as by chromatography [ 28 ]. Thus, the methods of HPLC and electrophoresis in polyacrylamide gel can be interchanged. This approach can also be proposed for monitoring product purification in various bioengineering processes (novel forms of food products [ 24 , 29 ] or novel sorbents for protein purification [ 30 ]) and in the development of pharmaceuticals [ 31 ].

Therefore, this approach can be employed for manufacturing pharmaceutically important products, such as bovine serum albumin. As a result, simultaneous quantitative and qualitative monitoring of purification of the target product, albumin, will be useful in novel technologies [ 30 ].

The patent for an invention of a method for antibody isolation and purification can be mentioned as an example of using this technique for pharmaceutical products [ 31 ]. In this and similar studies, it is also convenient and efficient to perform manufacturing process monitoring and simultaneous quantitative assessment of concentrations of immunoglobulin components both during the purification stages and in the target products using PAGE.

The applicability of protein quantification by electrophoresis is currently limited by the following factors [ 32 ]. Firstly, there are certain difficulties related to obtaining digital images of the gels. The currently available scanners and densitometers are not common equipment; their resolution is insufficient to work with a densitogram like with a chromatogram. Secondly, the existing software mostly specializes in electrophoresis of nucleic acids and therefore uses a different signal-to-noise ratio [ 33 ].

This study makes a methodological attempt to use electrophoresis for protein quantification. The specially designed test bench for digital imaging of gels and optimally selected software allows one to quickly and easily determine the molecular weight distribution of protein molecules in the samples and perform a quantitative assay. This will enable quality control of protein products according to the quantitative contents of fractions of protein molecules and the presence of impurities. The software allows, if necessary, to calculate complex protein samples with diffuse (blurred) protein bands and to exclude unwanted, useless bands on the gel from the calculations. So, the processing of gels allows to obtain more information than other methods: the qualitative and quantitative composition of protein mixtures, as well as their molecular weight distribution.

2. Materials and Methods

Materials. Bovine serum albumin (BSA; #SLBB7759V, Sigma Aldrich, St. Louis, MO, USA) was used as the model study object. This protein was applied both in its non-modified form and after vigorous mechanical treatment. A protein-containing product, Kultlab Isolate ISO 90% sports nutrition supplement (Kultlab, Novosibirsk, Russia) with 90% casein content, was used as an experimental study object.

The following reagents were also used for electrophoresis analysis in polyacrylamide gel: acrylamide (Sigma Aldrich, St. Louis, MO, USA), SDS (Sigma Aldrich, St. Louis, MO, USA), N,N,N’,N’-tetramethylethylenediamine (Sigma Aldrich, St. Louis, MO, USA), glycine (Sigma Aldrich, St. Louis, MO, USA), tris base (Sigma Aldrich, St. Louis, MO, USA), ammonium persulfate (Sigma Aldrich, St. Louis, MO, USA), dithiothreitol (Sigma Aldrich, St. Louis, MO, USA), glycerol (Sigma Aldrich, St. Louis, MO, USA).

Mechanical treatment. In order to obtain samples characterized by various degrees of protein molecule destruction, BSA and the Kultlab sports nutrition supplement predominantly containing casein were subjected to mechanical treatment on an AGO-2 laboratory planetary ball mill (acceleration of the grinding media, 200 m/s 2 ). Treatment duration was varied between 5 and 30 min. The weight of the sample loaded into the reaction jars was 5 g per 200 g of the grinding media (steel balls 6 mm in diameter).

SDS-polyacrylamide gel electrophoresis was carried out using the Laemmli protocol [ 20 ]. Polyacrylamide (Sigma Aldrich, St. Louis, MO, USA) concentration in the stacking and resolving gels was 5% and 13%, respectively. The gel was stained using Coomassie R-250 dye (Thermo Fisher Scientific, Waltham, MA, USA). An unstained protein MW marker (Thermo Fisher Scientific, Waltham, MA, USA) with protein molecular weight ranging between 14.4 and 116 kDa was used as a protein marker.

BSA solution in a lysing buffer (2 mg/mL) for being applied onto the gel lanes was prepared according to the Laemmli protocol [ 20 ]. The calibration BSA solutions were prepared by twofold serial dilution. BSA concentration in the calibration solutions ranged from 0.0125 to 0.2 mg/mL. For calibration, the solutions were applied in such a manner that BSA concentration on the polyacrylamide gel lanes was sequentially reduced twofold. The samples of protein mixtures (components of sports nutrition) after mechanochemical activation were prepared using the same procedure. Dilution of sports nutrition samples was selected so that the band intensity lay within the calibration plot.

Protein quantification. In order to save the electrophoresis results, photos of the gel were taken with a camera (Olympus, Tokyo, Japan) with a 64 MP resolution. The photos were taken on a specially designed test bench with six light sources ensuring uniform illumination of the object ( Figure 1 ).

A schematic diagram of the photographic test bench: ( A ) top view; ( B ) side view.

Taking photos of the gel under these conditions allows one to obtain a densitogram with a resolution being manifold higher than the resolution attainable using scanners for gels. It will be demonstrated below that densitograms can be processed in the same way as chromatograms.

The grey-tone photo images of polyacrylamide gels with stained protein bands were used for protein quantification. Mathematical data processing was performed using the Chrom & Spec software in order to obtain a dependence between protein concentration and band color intensity/peak area [ 34 , 35 , 36 ]. The results of quantitative measurements were processed and saved using the Chrom & Spec software (Ampersend Ltd., Moscow, Russia).

3. Results and Discussion

As already mentioned, the electrophoresis results are most often assessed visually, and it is a qualitative assessment. In this study, the results were analyzed using the Chrom & Spec software consisting of two programs: the Planar software for image conversion to densitograms and the Multi Chrom-Planar software performing quantitative processing of densitograms ( Figure 2 ) [ 34 , 35 ]. The area of the resulting chromatographic peaks in the densitogram depends on band color intensity, which corresponds to protein content. The calculations were performed for each peak according to the standard operating procedure of the Chrom & Spec software. A more detailed description of the technical part of the work in the software can be found in Refs. [ 34 , 36 ].

Example of converting the electrophoretic profile of a protein marker into a densitogram using the Chrom & Spec software.

Figure 3 shows an example of the electropherogram of calibration BSA samples prepared by serial dilution. One can see that band staining intensity in the solutions applied onto the gel varies in accordance with protein content ( Figure 3 ).

( A ) An example of the electropherogram of calibration BSA solutions with concentrations 0.2, 0.1, 0.05, 0.025, and 0.0125 mg/mL, respectively (lanes 1–5) and the reference sample with known molecular weights (lane 6). Volume of the applied sample = 10 µL. ( B ) A densitogram of the reference sample.

The electropherogram was converted to densitograms using the Chrom & Spec software ( Figure 3 B). As a result of data processing, the area of analytical peaks depending on band color intensity in the gel was measured for the BSA samples with different protein concentrations (lanes 1–5). Table 1 summarizes the results of intensity measurements (peak area in arb. units).

The resulting data for plotting the calibration plot.

The resulting data were used to build a calibration plot “protein concentration vs. peak area” for BSA (the calibration protein) ( Figure 4 ). A quadratic calibration dependence was obtained:

where Q is the protein content (µg), and S is the area of the chromatographic peak on the densitogram. This dependence is standard for planar chromatography or gel electrophoresis [ 37 , 38 ]. The relative deviation was 3.8%. The molecular weight of the analyzed bovine serum albumin (68.53 kDa) was determined using the known molecular weights of the protein marker. The results correlate with the UniProt database values [ 38 , 39 ]. For further studies, the calibration and test samples were applied to the same gel. In this case, all the calculations conducted for the same gel prevent the problems related to the possible non-uniformity of background staining and differences in gel concentration.

The calibration plot for BSA quantification.

In order to obtain BSA samples characterized by different degradation degrees, they were subjected to mechanical treatment for different times. Sample concentration for electrophoresis analysis was selected so as the intensity of the stained bands lay within the calibration curve plotted for native BSA (shown in the same gel on lanes 6–10) ( Figure 5 ).

( A ) Electropherogram of the BSA degradation products after mechanical treatment for 5, 10, 15, 20, and 30 min (lanes 1–5) and the calibration BSA samples with concentration ranging from 0.0125 to 0.2 mg/mL (lanes 6–10); volume of the applied sample = 10 µL. ( B ) Densitograms of the BSA degradation products after mechanical treatment for 5 and 30 min (lanes 1 and 5).

The calibration plot was used as the standard of quantitative measurements to calculate the amount of BSA remaining in the sample after mechanical treatment ( Figure 6 ). Figure 6 shows the data on the degree of degradation (α) of protein molecules calculated using the formula:

where ∆ S is the change in the area of the peak corresponding to the native protein molecule after mechanical treatment (treatment duration t); S t is the area of the peak corresponding to the native protein molecule after mechanical treatment (treatment duration t); and S 0 is the area of the peak corresponding to the native protein molecule before mechanical treatment. Protein molecules in BSA subjected to 30-min mechanical treatment were degraded by 92 ± 3%.

Protein content in the samples of mechanically treated BSA.

The experiment involving mechanical treatment of milk protein isolate (a sports nutrition mix) and quantitative calculation of casein degradation products during this treatment was conducted in a similar way. Figure 7 shows the electropherogram of the samples of milk protein isolate before and after mechanical treatment for 5, 10, 15, 20, and 30 min. One can see that the destruction of casein protein molecules also takes place during mechanochemical treatment ( Figure 7 ).

( A ) Electropherogram of the initial sample of milk protein isolate (lane 1) and the samples of milk protein after mechanical treatment for 30, 20, 15, 10, and 5 min, respectively (lanes 2–6), as well as the calibration BSA samples with concentration ranging from 0.125 to 2.0 mg/mL (lanes 7–10). ( B ) Densitograms of BSA degradation products after mechanical treatment for 5 and 30 min (lanes 2 and 6).

The BSA calibration plot was used to obtain a dependence that allowed one to calculate casein content in the samples (in µg) before and after mechanical treatment for 5, 10, 15, 20, and 30 min. The results are shown in Figure 8 . It was demonstrated that the degree of degradation of protein molecules within the sports nutrition product after mechanical treatment for 30 min was 85 ± 2%, being comparable to the data for an individual protein (BSA). The diffusivity (polydispersity) of protein bands as a result of mechanical processing is also noted. This effect is observed in the case of casein proteins, as well as to a large extent for pea proteins in previous research [ 23 ]. This circumstance was an important factor for choosing the method of quantitative calculation of protein in the gel, namely, the choice of the Chrom & Spec software.

Protein content in milk protein isolate before and after mechanical treatment for 5, 10, 15, 20, and 30 min.

For the purpose of industrial use of the methodology of quantitative calculation of milk proteins in the gel, a Thermo Fisher Scientific kit has been developed and is used. It includes the following equipment [ 40 ]. The procedure allows the quantification of the protein in the gel for various technological tasks, such as the control of protein impurities in dairy products, the regulation of the content of target protein substances with simultaneous control of the distribution of molecular weight. However, for unique research work, such a kit may be inconvenient, expensive, and unavailable. An application for smartphones has been developed for the express processing of gels, which allows the determination of the molecular weights of fragments of deoxyribonucleic acid or proteins with high accuracy [ 41 ]. This approach is very interesting because its use does not require specific equipment. However, this application does not allow quantifying analytes. The method proposed in this article allows you to quickly obtain information about the molecular mass distribution of molecules in the gel, as well as the quantitative and qualitative composition. Also, the Chrom & Spec software allows the use of not only standard operating procedures but to choose the appropriate mathematical processing independently, depending on the task set by the researchers. This requires enough standard equipment such as a personal computer with the necessary software and a camera (it is also possible to use a scanner or smartphone), so this approach is available to a large number of researchers working with electrophoresis.

Hence, it has been shown that polyacrylamide gel electrophoresis coupled with simultaneous recording photographic images of the gels and mathematical data processing using the Chrom & Spec software allows one to measure protein content in the test sample directly in polyacrylamide gel (identically to the known colorimetric methods for protein quantification). This technique has made it possible to estimate the degree of protein degradation for the model BSA protein and casein (a component of sports nutrition products). The procedure allows protein quantification for various applied problems such as performing control over protein impurities or regulating the content of target protein substances with simultaneous control over the molecular weight distribution.

4. Conclusions

In this work, we used photographic visualization of gels followed by mathematical data processing using Chrom & Spec software to quantify the intensity of protein bands in polyacrylamide gel. The results obtained proved that this algorithm can be used to process electrophoretic data and obtain accurate data regarding the quantitative analysis of proteins using this method. The relative inaccuracy of the method was estimated using calibration solutions of BSA. To make the protein quantification more accurate, it was proposed to use calibration solutions together with test samples on a single gel so that all variable factors could be taken into account during the analysis and recording of the results.

The possibilities of the presented method were tested on BSA and casein in the composition of a sports nutrition product, which were subjected to mechanical processing. The proposed method was used to obtain data on the relationship between the degree of degradation of protein molecules and the duration of mechanical processing. Mechanical treatment of BSA for 30 min resulted in degradation of protein molecules by 92 ± 3%, while protein molecules in a sports nutrition product were degraded by 85 ± 2%. The degree of destruction of an individual protein was higher compared to the degree of destruction of a protein-containing mixture after mechanical treatment for an identical period of time.

The methodological approach used in this study can serve as a guide for other researchers who would like to use electrophoresis to quantify protein both in individual form and in protein mixtures.

Author Contributions

Conceptualization, A.B.; funding acquisition, A.B.; investigation, K.D. and Z.A.; methodology, Z.A., A.B. and Y.K.; software, Y.K.; resources, A.B. and O.L.; writing—original draft preparation, K.D. and Z.A.; writing—review and editing, A.B., Y.K. and O.L.; visualization, K.D. All authors have read and agreed to the published version of the manuscript.

The research was funded within the state assignment to ISSCM SB RAS (project No. FWUS-2021-0005). Determination of energy consumption was carried out with the support of the Russian Science Foundation (project No. 19-73-10074).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Data availability statement, conflicts of interest.

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access is an initiative that aims to make scientific research freely available to all. To date our community has made over 100 million downloads. It’s based on principles of collaboration, unobstructed discovery, and, most importantly, scientific progression. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. How? By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers.

We are a community of more than 103,000 authors and editors from 3,291 institutions spanning 160 countries, including Nobel Prize winners and some of the world’s most-cited researchers. Publishing on IntechOpen allows authors to earn citations and find new collaborators, meaning more people see your work not only from your own field of study, but from other related fields too.

Brief introduction to this section that descibes Open Access especially from an IntechOpen perspective

Want to get in touch? Contact our London head office or media team here

Our team is growing all the time, so we’re always on the lookout for smart people who want to help us reshape the world of scientific publishing.

Home > Books > Biochemical Testing - Clinical Correlation and Diagnosis

Serum Protein Electrophoresis and Its Clinical Applications

Submitted: 10 April 2018 Reviewed: 03 July 2019 Published: 11 October 2019

DOI: 10.5772/intechopen.88367

Cite this chapter

There are two ways to cite this chapter:

From the Edited Volume

Biochemical Testing - Clinical Correlation and Diagnosis

Edited by Varaprasad Bobbarala, Gaffar Sarwar Zaman, Mohd Nasir Mohd Desa and Abdah Md Akim

To purchase hard copies of this book, please contact the representative in India: CBS Publishers & Distributors Pvt. Ltd. www.cbspd.com | [email protected]

Chapter metrics overview

2,827 Chapter Downloads

Impact of this chapter

Total Chapter Downloads on intechopen.com

Total Chapter Views on intechopen.com

This chapter focuses on the principle of electrophoresis and its utilization in a clinical laboratory. A sincere attempt has been made to discuss about clinical applications of serum protein electrophoresis, throwing light on the significance of serum protein electrophoresis in the management of multiple myeloma. Emphasis has been made on quality assurance in terms of accuracy and precision in electrophoresis to ensure reliability of patient results. A note on issues with lack of standardization of reporting of electrophoresis and an insight into global efforts to standardize the reporting of the assay has been included in this chapter.

• electrophoresis
• gamma globulins
• oligoclonal

Author Information

Satish ramanathan *.

• Division of Clinical Biochemistry, MIOT Hospitals, India

Chakravarthy Narasimhachar Srinivas

• Laboratory Medicine, MIOT Hospitals, India

*Address all correspondence to: [email protected]

1. Introduction

Serum protein electrophoresis is an electrophoretic method of separating proteins present in the serum to various fractions based on their molecular weight and electric charges. Electrophoresis had been widely used in clinical medicine for aiding in diagnosis of various clinical conditions like acute and chronic inflammations, monoclonal gammopathies, nephropathy, liver diseases, etc. This chapter discusses the clinical applications of serum protein electrophoresis [ 1 ] including the quality control practices and its implications [ 2 ].

2. Principle

The separation of proteins by electrophoresis is based on the fact that charged molecules usually migrate through a matrix/medium upon application of an electrical field [ 3 ]. The rate at which proteins move in an electric field is determined by a number of factors of the electrophoretic system and the nature of proteins itself. Some factors to mention are the strength of the electric field, temperature of the system, pH of the ions, concentration of buffer etc. [ 4 ]. Proteins vary in their size and shape and have the charges determined by the dissociation contents of their amino acids. Smaller proteins usually migrate faster, and larger proteins take a longer time. This physical property of proteins is exploited for its separation by employing the electrophoretic technique.

The use of “electroendosmosis” principle which improves the resolution of separation

Employing a “high-voltage” electric current which aids in improving the throughput (the processing time) and the resolution of protein separation.

Below is an illustration of capillary electrophoresis (Sebia Minicap Flex Piercing) ( Figure 1 ). Sebia Minicap Flex Piercing capillary electrophoresis works on the principle of capillary electroendosmosis under high-voltage electric current. The Flex Piercing model of Sebia CZE aids in testing of human blood with capped tubes which in turn eliminates the biohazard xassociated with handling of uncapped samples.

Sebia Minicap flex piercing capillary electrophoresis.

3. Revisiting the basics: an insight into the protein family

Serum proteins are a family of albumin and globulins. Albumin is the major fraction synthesized from human liver endogenously and available through various dietary sources exogenously including egg, meat, pulses, milk etc. Globulins are a group of proteins subclassified into alpha-1, alpha-2, beta-1, beta-2, and gamma globulins based on the electrophoretic mobility ( Figure 2 ). The normal biological interval of serum total proteins in a healthy adult ranges between 6 and 8 g/dl which includes Serum Albumin: 3.5–4.5 g/dl and Globulins: 2.5–3.5 g/dl.

Serum protein family with fractions.

3.1 Albumin

Albumin is a 69 kDa protein. It is the most abundant protein in serum. Albumin is synthesized in the liver and functions as a transport protein of various substances like bilirubin, enzymes, hormones, drugs etc. It also maintains fluid volume within the vascular space. Albumin is the first protein fraction to appear near the anode in SPE. Altered levels of serum albumin are associated with various clinical conditions. Low levels of albumin are clinically significant and are termed as hypoalbuminemia.

Decreased concentration of serum albumin (hypoalbuminemia) indicates either a poor dietary intake (malnutrition) or a decreased production or an increased loss. Chronic liver disease is a common clinical condition associated with decreased albumin production, and chronic kidney disease (CKD) is the most common disease associated with an increased loss of albumin in urine (proteinuria). This clinical condition is otherwise known as nephropathy. Other causes of hypoalbuminemia include acute and chronic inflammation, critical illness, pregnancy etc.

Bilirubin, Triglycerides if present in high levels in serum may appear as a blunt peak which is seen adjacent to the cathode near the albumin peak.

Prealbumin (transthyretin)—increased levels of pre albumin, if present due to various clinical conditions including several inflammatory diseases is seen as a blunt anodal peak distinctly separated from the peak of albumin.

A rare variant observed in the albumin peak is bisalbuminemia which is a rare condition, with no clinical features, in which the serum contains two albumin variants of different electrophoretic mobilities, usually in equal concentrations, though the total concentration of albumin is normal. Bisalbuminemia may be hereditary or acquired. The acquired type has been more frequently reported in chronic renal disease and pancreatitis and in patient with chronic renal disease. Two (rather than one) albumin bands may represent bisalbuminemia. Hereditary condition is a rare anomaly caused by a genetic lesion in the albumin gene usually a point mutation.

Analbuminemia (absence of albumin) is another genetically inherited metabolic disorder and was first described in 1954. This disorder is rare and affects less than 1 in 1 million births.

The most important aspect of such albumin variants lies in quantification of an albumin peak in such scenarios followed by interpretation and clinical correlation ( Figure 3 ).

Abnormal electrophoretic patterns of albumin zone.

3.2 Alpha fraction

As electrophoresis proceeds toward the negative portion of the gel (cathode), the alpha zone is the next band after albumin. The alpha zone is subdivided into two zones: the alpha-1 peak and alpha-2 peak.

The alpha-1 peak consists of alpha-1 antitrypsin (AT), alpha-1-chymotrypsin, and thyroid-binding globulin. Alpha-1 antitrypsin is an acute-phase reactant. The concentration of alpha-1 antitrypsin increases in conditions of inflammation and is usually decreased in patients with alpha-1 antitrypsin deficiency or decreased production of globulin in patients with severe liver disease. A rare variant of alpha-1 antitrypsin is encountered occasionally characterized by a split peak pattern of alpha-1 globulins.

The alpha-2 peak consists of alpha-2 macroglobulin, haptoglobin, and ceruloplasmin. Alpha-2 macroglobulin accounts for about 3% of the total protein in the serum. Because of the variable migration of the haptoglobin types, a2-macroglobulin is often adjacent to, or co migrating with, haptoglobin and is therefore not seen as a discrete band.

A distorted pattern of alpha-2 region in electrophoresis is seen commonly in conditions of hemolysis, including in vivo and in vitro. The pathophysiology behind this pattern is the formation of hemoglobin-haptoglobin complexes in these conditions. This is a physiological adaptive response by human physiology to conserve hemoglobin released as a result of RBC breakdown into circulation and hemoglobin being a smaller globular protein is bound to be lost in urine. Hence to preserve it, haptoglobin is consumed to form complex with hemoglobin which results in the formation of a macromolecular protein which is retained in circulation making hemoglobin available for the production of RBCs and prevention of anemia.

Haptoglobin and ceruloplasmin are acute-phase reactants, and hence increased in acute inflammatory states.

Alpha-2-macroglobulin is increased in nephrotic syndrome and cirrhosis of the liver. The elevation of alpha-2 macroglobulin is distinctly evident in nephritic syndrome, since it is a bulky molecule, and hence retained in circulation to compensate for the loss of other proteins in urine which is evident in form of proteinuria in urine microscopic examination.

Ceruloplasmin is an important copper-binding transport protein produced by the liver. Ceruloplasmin concentrations are markedly decreased in conditions of Wilson’s disease. The disadvantage of serum protein electrophoresis is that it will not aid in the detection of a decreased ceruloplasmin.

3.3 Beta fraction

The beta zone usually is subdivided into two peaks, beta-1 and beta-2 in CZE. Beta-1 zone comprises proteins like transferrin and low-density lipoprotein (LDL).

Transferrin functions to transport non-heme ferric iron from the gastrointestinal tract. Each Transferrin molecule can bind two molecules of free iron. An increased beta-1 band is observed in iron deficiency anemia due to an increased level of free transferrin and also in pregnancy. Determinations of the transferrin levels are useful in distinguishing between iron deficiency anemia (inadequate intake or chronic hemorrhage with loss of iron stores) and hemolytic anemia, in which transferrin levels are low resulting in a beta-1 peak of low amplitude. Transferrin is usually decreased in alcoholic cirrhosis. Transferrin is also decreased during renal disease and thermal injuries.

The beta-2 band is mostly composed of complement proteins, C3 and C4. Elevated beta-2 zone can be caused in inflammatory states due to activation of complement cascade which include C3 and C4 too.

A reduced beta-2 peak intensity can be encountered in an aged sample, since the immune complexes are used up and low serum levels of complements are evidenced.

Fibrinogen is a protein with molecular weight of 340 kDa protein. Sometimes a small fibrinogen band can be seen in serum protein electrophoresis due to the insufficient clotting or failure to remove the serum from the clot. This fibrinogen band is seen between beta-1 and beta-2 regions. This band is also seen in patients who are receiving heparin therapy. It is also an important indicator of the sample type being analyzed. When plasma is used in the place of serum for protein electrophoresis, fibrinogen present in plasma appears in the beta-2 region, and this has the potentiality to interfere with the detection of monoclonal gammopathies in such patients ( Figure 4 ).

Fibrinogen producing a peak in beta 2 region (from a plasma sample).

3.4 Gamma fraction

One of the main clinical implications of serum protein electrophoresis is to aid diagnosis of disorders associated with alterations of gamma globulins. Gamma region comprises mainly of serum immunoglobulins. The five major classes of immunoglobulins are IgG, IgA, IgM, IgD, and IgE. The immunoglobulins are characterized by the presence of two protein moieties named as heavy chain and light chain. The classification of immunoglobulin had been made based on the composition of heavy chains, while the light chains are of two types including kappa or lambda. Physiologically, kappa forms the major light chain fraction among the two.

Hypergammaglobinemia (increased serum gamma globulin levels)

Hypogammaglobinemia (decreased serum gamma globulin levels)

Hypergammaglobinemia (gammopathies):

Gammopathy is defined as abnormal proliferation of the lymphoid cells producing immunoglobulins. There are four types of gammopathies: polyclonal, monoclonal, biclonal, and oligoclonal.

Polyclonal gammopathies are defined as heterogeneous increase in immunoglobulins involving more than one cell line, commonly caused by a variety of inflammatory conditions (chronic inflammation), infections, chronic liver diseases (cirrhosis), chronic kidney diseases, etc.

Monoclonal gammopathies are characterized by a homogenous increase produced by clonal population of mature B cells, most commonly plasma cells. Monoclonal immunoglobulins seen in these conditions are also known as Para proteins. The classic interpretative terminology used in clinical laboratory medicine for describing a monoclonal immunoglobulin in SPE is “M” band where M stands for monoclonal. Common clinical disorders producing “M” Band in SPE include multiple myeloma and plasmacytoma in usually 60% of cases and Waldenströms Macroglobulinemia, lymphomas, and leukemia in approximately 10% of cases. Certain monoclonal gammopathies produce “M” band in electrophoretic regions other than in gamma regions, commonly being beta region especially in case of IgA and IgG myeloma.

Biclonal gammopathies are characterized by a double peak in the gamma region. This electrophoretic pattern is seen when there is a biclonal proliferation of immunoglobulins encountered in multiple myeloma. A biclonal pattern is also seen in monoclonal gammopathies associated with IgA and IgG. In such scenarios, these immunoglobulins appear as polymerized and monomerized forms which elute as biclonal peaks in gamma region or in beta region, respectively ( Figure 5 ).

Abnormal electrophoretic patterns of gamma zone.

The oligoclonal pattern of gamma region is characterized by more than two peaks evident in the gamma region. This pattern is commonly seen in autoimmune disorders, light chain myelomas (characterized by clonal proliferation of light chains), amyloidosis, etc. ( Figure 5 ).

Apart from serum immunoglobulin, C-reactive protein (CRP) also is evident in the gamma region. C-reactive proteins levels usually increase during inflammatory responses.

Apart from the common causes of altered electrophoresis picture specific to the particular zones, a sharp distinct peak when evident especially in beta or alpha region should raise a high index of diagnostic suspicion of multiple myeloma since a few monoclonal immunoglobulins shall migrate in these zones too, in contrary to the classical gamma zone M protein pattern, which is commonly reported in these conditions.

4. Role of SPE in multiple myeloma work-up

MGUS—monoclonal gammopathy of undetermined significance

MGRS—monoclonal gammopathy of renal significance

Smoldering myeloma

Multiple myeloma (which includes various subtypes including nonsecretory myeloma (NSMM), light chain myeloma, secretory multiple myeloma)

Monoclonal gammopathy of undetermined significance (MGUS)

M protein (Monoclonal band)—<3 g/dl

Bone marrow biopsy—<10% plasma cells seen

No clinical symptoms/signs

Normbal free light chain ratio in serum

Monoclonal gammopathy of renal significance (MGRS)

M protein (monoclonal band)—<3 g/dl

Renal disease with elevated serum creatinine

Normal Free light chain ratio in serum

Bone marrow biopsy—>10% plasma cells seen

Abnormal free light chain ratio in serum

Clinically significant. Clinical diagnosis includes a tetrad of “ÇRAB” which stands for (one of the four shall be present):

C—hypercalcemia

R—renal abnormalities (elevated creatinine)

A—anemia

B—bone lesions

Multiple myeloma

M protein (monoclonal band)—>3 g/dl

Clinical diagnosis includes a tetrad of “ÇRAB” which stands for (one of the four shall be present):

A —anemia

There are exceptions in SPE findings in certain cases of multiple myeloma wherein the SPE does not reveal any significant alteration or a clue toward the diagnosis.

These variants of multiple myeloma characterized by an abnormal bone marrow (increased plasma cells) but a normal SPE are termed as nonsecretory myelomas which account to 1–2% of multiple myelomas. In such cases, an immunoassay of free light chains (FLC) in serum provides a diagnostic clue toward NSMM which show a significant disproportionate elevation of usually a clone of light chains (kappa or lambda) with an alteration in kappa/lambda ratio (normal Ratio is between 0.60 and 1.65). A commonly encountered phenomenon with laboratory testing of FLC includes “prozone” effect or “hook” effect which occurs due to antigen excess and requires appropriate dilution to obtain reliable results.

Bence-Jones protein estimation in urine is an antique piece of laboratory evidence toward multiple myeloma, which is characterized by detection of light chains in urine. But since the methodology of testing is manual and does not provide standardization, this has been replaced by urine FLC analysis in laboratories practicing good clinical laboratory practices (GCLP).

One more valiant laboratory investigation which is an essential requisite for multiple myeloma work-up includes immunoelectrophoresis.

One common principle employed in immunoelectrophoretic technique involves the use of specific antihuman immunoglobulins (e.g., Anti-IgG, Anti-IgA, Anti-Kappa, etc.) as a preprocessing step which results in precipitation of immunoglobulins if present and disappearance of the band/peak contributed by that specific immunoglobulin. Hence this technique is also known as immunosubtraction. This technique aids in typing the specific type of immunoglobulin (including the type of light chain) contributing to myeloma. This technique is supplemented by quantification of serum immunoglobulins by an immunoassay.

4.1 SPE and its clinical significance

SPE is a semiquantitative investigation which involves technical expertise to recognize the specific electrophoretic patterns and associate with various clinical conditions. This requires a laboratory practice integrated across various divisions of laboratory and with respective clinical and ancillary divisions of clinical medicine [ 1 ].

With respect to SPE, the laboratory professionals shall act as consultants to the clinical consultants. This is possible in scenarios where the clinician does not arrive at a provisional diagnosis of a gammopathy and the laboratory picks up the diagnostic clue toward gammopathy through an increased serum total protein level (<8 g/dl) and an altered serum albumin globulin (AG) ratio (which is usually altered in gammopathy). A normal AG ratio ranges between 1.2 and 1.8, while there is a significant reduction in the ratio in patients with gammopathy. This becomes an incidental finding which leads to a concept of “reflex” testing for multiple myeloma work-up including SPE, upon consent from the treating clinician and the patient.

Inflammation: Increased intensity of alpha-1 and alpha-2 with a sharp leading edge of alpha-1 may be observed, but with chronic inflammation the albumin band may be decreased with increased gamma zone due to the polyclonal gammopathy.

Nephrotic syndrome: The albumin band is decreased due to hypoalbuminemia. In addition, the alpha-2 band may be more distinct.

Cirrhosis or chronic liver disease: A low albumin band due to significant hypoalbuminemia with a prominent beta-2 band and beta-gamma bridging is a characteristic feature. In addition, polyclonal hypogammaglobinemia is observed.

Malnutrition: Decreased albumin levels [ 1 ].

Alpha-1 antitrypsin deficiency : Inflammatory condition, pregnancy.

Hemolysis: Altered electrophoretic pattern of small indistinct peaks in alpha-2 region.

4.2 Quality assurance in SPE

Quality assurance in SPE is an essential prerequisite to ensure reliability of an SPE result [ 2 ]. There are two major aspects of analytical quality including precision (measure of precision) and accuracy (measure of trueness).

Good clinical laboratory practices demand processing of an internal quality control (IQC) for assessment of precision and external quality assurance (EQA)/proficiency testing (PT testing) for accuracy assessment. IQC is a material which can be prepared in house (patient sample) or available commercially and is to be processed before a patient sample is taken up for processing.

The clinical laboratory has its responsibility to select and use an IQC which has a matrix comparable to patient sample, preferably covering the clinical decision point (cut off value that differentiates between a normal and abnormal result). EQA is an external assessment of the analytical quality wherein the laboratory processes a blinded sample and the results are compared against a reference method and/or against the consensus value of other participant laboratories for that specific sample.

The laboratory has to hold responsibility in selecting a suitable EQA provider who shall preferably be accredited to ISO 17043. If an EQA program is not available, the laboratories shall participate in exchange of samples with referral laboratories with a similar methodology and a comparable quality of testing standard.

5. Reporting of results and its standardization

Reporting SPE requires interpretation of the electrophoretic pattern which is followed by comments of such an interpretation along with the piece of advice to the clinician if indicated. There is a big lacuna in the format of reporting of SPE, each laboratorian using his/her own means of interpreting and communicating. It is the need of the hour to have a standardized format of reporting SPE for ensuring patient safety and clinician follow-up. There are no international guidelines, though the working party on standardized reporting of protein electrophoresis which is an initiative of the Australasian Association of Clinical Biochemists has come out with a standardized format of reporting SPE.

6. Conclusion

In the current scenario, it becomes the responsibility of each and every laboratory to ensure that all relevant information is available in a SPE report, easily read, understood, and interpreted by a clinician. This becomes the core of a clinical laboratory practice.

Acknowledgments

We would wish to acknowledge and thank the management of MIOT hospitals for providing us with the infrastructure and technology to explore, learn, and contribute to our patients’ well-being.

We would wish to thank our technical staff, Mr. Mathivanan Durairaj, and his team for their invaluable contribution by sharing clinical cases.

We wish to acknowledge Trivitron technologies for their valuable support in installation and continual service and application support for Sebia CZE.

• 1. O’Connell TX, Horita TJ, Kasravi B. Understanding and interpreting serum protein electrophoresis. American Family Physician. 2005; 71 (1):105-112
• 2. Jenkins MA. Electrophoresis. Quality Control and Quality Assurance Aspects of the Routine use of Capillary Electrophoresis for Serum and Urine Protein in Clinical Laboratories. Wiley Online Library. Jun 2004; 25 (10-11):1555-1560
• 3. Jenkins MA. Serum Protein Electrophoresis. Clinical Applications of Capillary Electrophoresis. Molecular Biotechnology; 2000. pp. 11-19
• 4. Ninfa AJ, Ballou DP, Benore M. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Hoboken, NJ: Wiley; 2009. p. 161
• 5. Smith I, editor. Zone Electrophoresis: Chromatographic and Electrophoretic Techniques. 4th ed. Elsevier Ltd.; 1976
• 6. Tate J, Caldwell G, Daly J, Gillis D, Jenkins M, et al. Recommendations for standardized reporting of protein electrophoresis in Australia and New Zealand. Annals of Clinical Biochemistry. 2012; 49 :242-256

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Continue reading from the same book

Biochemical testing.

Edited by Varaprasad Bobbarala PhD

Published: 29 April 2020

By Husniza Hussain, Rusidah Selamat, Lim Kuang Kuay, ...

2086 downloads

By Khushaboo Pandey and Om Prakash Mishra

1199 downloads

By Nataša Gros

1314 downloads

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals

Electrophoresis articles from across Nature Portfolio

Electrophoresis is the process by which large charged molecules travel through a medium under a uniform electric field. The most common implementation, gel electrophoresis, is a method used to separate nucleic acids by length or proteins by size, conformation and charge based on their migration through a porous matrix.

Latest Research and Reviews

Valproate regulates inositol synthesis by reducing expression of myo -inositol-3-phosphate synthase

• Kendall C. Case
• Rachel J. Beltman
• Miriam L. Greenberg

Targeted profiling of human extrachromosomal DNA by CRISPR-CATCH

CRISPR-CATCH is used to isolate extrachromosomal DNA (ecDNA) molecules containing oncogenes from human cancer cells. CRISPR-CATCH followed by nanopore sequencing allows for methylation profiling, highlighting differences from the native chromosomal loci.

• King L. Hung
• Jens Luebeck
• Howard Y. Chang

Beovu, but not Lucentis impairs the function of the barrier formed by retinal endothelial cells in vitro

• Heidrun L. Deissler
• Catharina Busch
• Matus Rehak

Nematode CDC-37 and DNJ-13 form complexes and can interact with HSP-90

• Lukas Schmauder
• Eva Absmeier
• Klaus Richter

A new humanized antibody is effective against pathogenic fungi in vitro

• Tomas Di Mambro
• Tania Vanzolini
• Mauro Magnani

Benchmarking laboratory processes to characterise low-biomass respiratory microbiota

• Raiza Hasrat
• Jolanda Kool
• Thijs Bosch

News and Comment

Recovery of intact DNA nanostructures after agarose gel–based separation

• Gaëtan Bellot
• Mark A McClintock
• William M Shih

Guidelines for reporting the use of gel image informatics in proteomics

• Christine Hoogland
• Martin O'Gorman
• Andrew R Jones

Guidelines for reporting the use of capillary electrophoresis in proteomics

• Paula J Domann
• Satoko Akashi
• Chris F Taylor

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Two-Dimensional Electrophoresis: An Overview

Series: Methods In Molecular Biology > Book: Two-Dimensional Electrophoresis Protocols

Overview | DOI: 10.1007/978-1-59745-281-6_1

• Department of Biology, McMaster University, Room 208/203, Life Sciences Building, 1, Hamilton, ON, Canada

Two-dimensional gel electrophoresis (2DE) separates proteins by molecular charge and molecular size. Proteins are first solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent.

Two-dimensional gel electrophoresis (2DE) separates proteins by molecular charge and molecular size. Proteins are first solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent. First-dimension isoelectric keywords, focusing, then subjects proteins to a high voltage within a pH gradient. The amphoteric nature of proteins means each migrates to the pH where the net molecular charge is zero. After equilibration, to ensure complete protein unfolding, the second dimension separates by molecular size. Each protein is therefore resolved at a unique isoelectric point/molecular size coordinate. After visualisation by staining proteome changes are revealed by gel image analysis, and protein spots of interest excised and identified by mass spectrometry sequence analysis combined with database comparison. Variations to this procedure include staining or radio-labelling prior to electrophoresis. Although 2DE does have limitations, the most significant being the resolution of membrane and/or hydrophobic proteins, the potential solutions offered by pre-fractionation or adjustments to the electrophoresis regimen mean this technique is likely to remain central to proteomic research.

Figures ( 0 ) & Videos ( 0 )

Experimental specifications, other keywords.

Related articles

Dige analysis of human tissues, the basics of 2d dige, 2-d dige combined with pro-q diamond staining for the identification of protein phosphorylation for chlamydomonas reinhardtii : a successful approach, the latest advancements in proteomic two-dimensional gel electrophoresis analysis applied to biological samples, visible and fluorescent staining of two-dimensional gels, two-dimensional gel electrophoresis for the identification of signaling targets, two-dimensional gel electrophoresis in studies of flower and leaf proteome of common buckwheat, two-dimensional gel electrophoresis with immobilized ph gradients, two-dimensional gel electrophoresis: vertical isoelectric focusing, 2d-dige analysis of eye lens proteins as a measure of cataract formation.

• Stochaj, W. R., Berkelman, T., and Laird, N. (2003) Preparative two-dimensional gel electrophoresis with immobilised pH gradients, in Proteins and Proteomics, A Laboratory Manual (Simpson, R. J., ed.), Cold Spring Harbor, New York, pp. 143–218
• Graves, P. R., and Haystead, T. A. (2003) Proteomics and the Molecular Biologist. In Handbook of Proteomic Methods (Conn, P. M., ed.), Humana, New Jersey, pp. 3–16
• Speicher, D. W. (2004) Overview of Proteome Analysis. In Proteome analysis. Interpreting the Genome (Speicher, D. W., ed.), Elsevier, Amsterdam, pp. 19–73
• Pandey, A., and Mann, M. (2000) Proteomics to study genes and genomes. Nature 405, 837–846
• Anderson, L., and Seilhamer, J. (1997) A comparison of selected mRNA and proteins abundance in human liver. Electrophoresis 18, 53–537
• Gygi, S. P., Rochon, Y., Franza, B. R., and Abersold, R. (1999) Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19, 1720–1730
• Godley, A. A., and Packer, N. M. (1997) The Importance of protein co-and post-translational modifications in Proteome Projects. In Proteome Research: New Frontiers in Functional Genomics (Wilkins, M. R., Williams, K. L., Appel, R. D., and Hochstrasser, D. F., eds.), Springer, Berlin, pp. 65–91
• O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007–4021
• Klose, J. (1975) Protein mapping by combined isoelectric focussing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik 26, 231–243
• Fichmann, J., and Westermeier, R. (1998) 2-D protein gel electrophoresis. An overview. In Methods in Molecular Biology, Vol 112: 2-D Proteome Analysis Protocols (Link, A. J., ed), Humana Press inc, New Jersey
• Görg, A., Drews, O., and Weiss, W. (2004) Separation of proteins using two-dimensional electrophoresis, in Purifying Proteins for Proteomics, A Laboratory Manual (Simpson, R. J., ed.), Cold Spring Harbor, New York, pp. 391–430
• Görg, A., and Wiess, W. (2004) Protein profile comparisons of microorganisms, cells and tissues using 2D gels, in Proteome Analysis . Interpreting the Genome (Speicher, D. W., ed.), Elsevier, Amsterdam, pp. 19–73
• Twyman, R. M. (ed.) (2004) Principles of Proteomics. Garland Science / Bios scientific publishers. New York.
• Rabilloud, T. (1999) Solubilization of proteins in 2-D electrophoresis. An outline. In Methods in Molecular Biology, Vol 112: 2-D Proteome Analysis Protocols (Link, A. J., ed.), Humana Press inc, NJ.
• Dunn, M. J. (1987) Two-dimensional gel electrophoresis, in Advances in Electrophoresis, Vol 1 (Chrambach, A., Dunn, M. J., and Radola, B. J., eds.), VCH Weinheim, New York, pp. 1–109
• Dunn, M. J. (1993) Gel Electrophoresis: Proteins . BIOS, Oxford.
• Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. (1997) Improvements of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18, 307–316
• Musante, L., Candiano, G., and Chiggeri, G. M. (1997) Resolution of fibronectin and other uncharacterised proteins by two-dimensional polyacrylamide electrophoresis with thiourea. J. Chromatogr. 705, 351–356
• Perdew, G. H., Schaup, H. W., and Selivonchick, D. P. (1983) The use of a zwitterionic detergent in two-dimensional gel electrophoresis of trout liver microsomes. Anal. Biochem. 135, 435–455
• Adessi, C., Miege, C., Albrieux, C., and Rabilloud, T. (1997) Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients. Electrophoresis 18, 127–135
• Harder, A., Wildgruber, R. Nawrocki, A., Fey, S. J., Larsen, P. H., and Görg, A. (1999) Comparison of yeast cell protein solubilization procedures for two-dimensional electrophoresis. Electrophoresis 20, 826–829
• Herbert, B. R., Molloy, M. P., Gooley, A. A., Walsh, B. J., Bryson, W. G., and Williams, K. L. (1998) Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as the reducing agent. Electrophoresis 19, 845–851
• Rabilloud, T., Valette, C., and Lawrence, J. J. (1994) Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15, 1552–1558
• Guy, C. R., Philip, R., and Tan, Y. H. (1994) Analysis of cellular phosphoproteins by two-dimensional electrophoresis. Applications for cell signalling in normal and cancer cells. Electrophoresis 15, 417–440
• Flengsrud, R., and Kobro, G. (1989) A method for two dimensional electrophoresis of proteins from green plant tissue. Anal. Biochem. 177, 33–36
• Taylor, R. S., Wu, C. C., Hays, L. G., Eng, J. K.,Yates, J. R. I., and Howell, K. E. (2000) Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation. Electrophoresis 21, 3441–3459
• Damerval, C., de Vienne, D., Zivy, M., and Thiellement, H. (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis 7, 52–54
• Görg, A., Obermaier, C., Boguth, G., Csordas, A., Diaz, J. J., and Madjar, J. J. (1997) Very alkaline immobilized pH gradients for two-dimensional electrophoresis of ribosomal and nuclear proteins. Electrophoresis 18, 328–337
• Görg, A. (1999) IPG-Dalt of very alkaline proteins, in Methods in Molecular Biology: 2-D Proteome Analysis Protocols, Vol 112, pp. 197–209 (Link, A. J., ed.), Humana, New Jersey
• Wildgruber, R., Reil, G., Drews, O., Parlar, H., and Görg, A. (2002) Web-based two-dimensional database of Saccharomyces cerevisiae proteins using immobilized pH gradients from pH 6 to pH 12 and matrix-assisted laser desorption/ionization – time of flight mass spectrometry. Proteomics 2, 727–732
• Görg, A., Boguth, G., Obermaier, C., and Weiss, W. (1998) Two-dimensional electrophoresis of proteins in an immobilized pH 4–12 gradient. Electrophoresis 19, 1516–1519
• Olivieri, E., Herbert, B., and Righetti, P. G. (2001) The effect of protease inhibitors on the two-dimensional electrophoresis pattern of red blood cell membranes. Electrophoresis 22, 560–565
• Grainer, F. (1988) Extraction of plant proteins for two-dimensional electrophoresis. Electrophoresis. 9, 712–718
• Scheele, G. A. (1975) Two-dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. J. Biol. Chem. 250, 5375–5385
• Bjellqvist, B., Ek, K., Righetti, P. G., Gianazza, E.,Görg, A., Westmeier, R., and Postel, W. (1982) Isoelectric focussing in immobilized pH gradients: principles, methodologies and some applications. J. Biochem. Biophys. Methods 6, 317–339
• Görg, A., Postel, W., and Günther, S. (1985) Improved horizontal two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 6, 599–604
• Görg, A., Postel, W., and Günther, S. (1988) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 9, 531–546
• Corbett, J. M., Dunn, M, J., Posch, A., and Görg, A. (1994) Positional reproducibility of protein spots in two-dimensional polyacrylamide gel electrophoresis using immobilised pH gradient isoelectric focussing in the first dimension: an interlaboratory comparison. Electrophoresis 15, 1205–1211
• Blomberg, A., Blomberg, L., Norbeck, J., Fey, S. J., Larsen, P. H., Roepstorff, P., et al. (1995) Interlaboratory reproducibility of yeast protein patterns analysed by immobilized pH gradient two-dimensional gel electrophoresis. Electrophoresis 16, 1935–1945
• Righetti, P. G., and Bossi, A. (1997) Review. Isoelectric focusing in immobilized pH gradients: an update. J. Chromatogr. B, 699, 77–89
• Righetti, P. G., Gianazza, E., and Bjellqvist, B.(1983) Minireview. Modern aspects of isoelectric focusing: two-dimensional maps and immobilized pH gradients. J. Biochem. Biophys. Methods 8, 8–108
• Bjellqvist, B., Sanchez, J. C., Pasquali, C., Ravier, F., Paquet, N., Frutiger, S., et al. (1993) Micropreparative two-dimensional electrophoresis allowing the separation of samples containing milligram amounts of proteins. Electrophoresis 14, 1375–1378
• Görg, A., Obermaier, C., Boguth, G., Harder, A.,Scheibe, B., Wildgruber, R., and Weiss, W. (2000) The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 21, 1037–1234
• Görg, A. (1991) Two-dimensional electrophoresis. Nature 349, 545–546
• Görg, A., Postel, W., Weser, J., Günther, S., Strahler, S. R., Hanash, S. M., and Somerlot, L. (1987) Elimination of point streaking on silver stained two-dimensional gels by addition of iodoacetamide to the equilibration buffer. Electrophoresis 8, 122–124
• Zou, X., and Speicher, D. W. (2002) Comprehensive analysis of complex proteomes using microscale solution isoelectric focussing prior to narrow pH range two-dimensional electrophoresis. Electrophoresis 2, 58–68
• Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
• Weber, K., and Osborne, M. (1969) The reliability of molecular weight determination by dodecyl sulphate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412
• Shägger, H., and von Jagow, G. (1987) Tricine-sodium dodecyl-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 1000 kDa. Anal. Biochem. 166, 368–379
• Rabilloud, T. (2000) Detecting proteins separated by 2-D gel electrophoresis. Anal. Chem. 72, 48A–55A
• Rabilloud, T., and Charmont, S. (2000) Detection of proteins on two-dimensional electrophoresis gels. In Proteome Research: Two Dimensional Electrophoresis and Identification Methods (Rabilloud, T., ed.), Springer, Berlin, pp. 107–126
• Patton, W. F. (2002) Detection technologies in proteome analysis. J. Chromatogr. B. Anal. Technol. Biomed. Life Sci. 771, 3–31
• Neuhoff, V., Arold, N., Taube, N., and Ehrhardt, W. (1988) Improved staining of proteins in polyacrylamide gels including isoelectric gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 9, 255–262
• Lopez, M. F., Berggren, K., Chernokalskaya, E.,Lazarev, A., Robinson, M., and Patton, W. (2000) A comparison of silver stain and SYPRO ruby protein gel stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis 21, 3673–3683
• Gatlin, C. L., Kleeman, G. R., Hays, L. G., Link, A. J., and Yates, J. R. III (1998) Protein identification at the low femtomole level form silver-stained gels using and new fruitless electrospray interface for liquid chromatography - microspray and nanospray mass spectrometry. Anal. Biochem. 263, 93–101
• Jensen, O. N., Mortensen, P., Vorm, O., and Mann, M. (1997) Automation of matrix-assisted laser desorption/ionization mass spectrometry using fuzzy logic feedback control. Anal. Chem. 69, 1706–1714
• Castellanos-Serra, L., Proenza, W., Huerta, V.,Moritz, R. L., and Simpson, R. L. (1999) Proteome analysis of polyacrylamide gel-separated proteins visualized by reversible negative staining using imidazole-zinc salts. Electrophoresis 20, 732–737
• Switzer, R. C., Merril, C. R., and Shifrin, S. (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal. Biochem. 98, 321–327
• Rabilloud, T., Vullard, L., Gilly, C., and Lawrence, J. J. (1994) Silver staining of proteins in polyacrylamide gels: a general overview. Cell. Mol. Biol. 40, 57–75
• Merril, C. R., Goldman, D., Sedman, S. A., and Ebert, M. H. (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211, 1437–1438
• Oakley, B. R., Kirsch, D. R., and Morris, N. R.(1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal. Biochem. 105, 361–363
• Shevenko, A., Wilm, M., Vorm, O., and Mann, M.(1996) Mass spectrometric sequencing of protein silver-stained polyacrylamide gels. Anal. Chem. 68, 850–858
• Mortz, E., Krogh, T. N., Vorum, H., and Görg, A. (2001) Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization time of flight analysis. Proteomics 1, 359–363
• Yan, J. X., Walt, R., Berkelman, T., Harry, R. A., Westbrook, J. A., Wheeler, C. H., and Dunn, M. J. (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray mass spectrometry. Electrophoresis 21, 3666–3672
• Scheler, C., Lamer, S., Pan, Z., Li, X., Salinkow, J., and Jungblut, P. (1998) Peptide mass fingerprint sequence coverage from differently stained proteins on two-dimensional electrophoresis patterns by matrix assisted laser desorption/ionization – mass spectrometry (MALDI-MS). Electrophoresis 19, 918–927
• Gharahdaghi, F., Weinberg, C. R., Meagher, D. A., Imai, B. S., and Mische, S. M. (1999) Mass spectrometric identification of proteins form silver-stained polyacrylamide gels: a method for the removal of silver ions to enhance sensitivity. Electrophoresis 20, 601–605
• Sinha, P., Poland, J., Schnölzer, M., and Rabilloud, T. (2001) A new silver staining apparatus and procedure for matrix/ionization – time of flight analysis of proteins after two-dimensional electrophoresis. Proteomics 1, 835–840
• Patton, W. F., Lim, M. J., and Shepro, D. (1999) Image acquisition in 2-D electrophoresis. Methods Mol. Biol. 112, 353–362
• Celis, J. E., and Gromov, P. (1999) 2D protein electrophoresis: can it be perfected? Curr. Opin. Biotechnol. 10, 16–21
• Sanchez, J-C., Chiappe, D., Converset, V., Hoogland, C, Binz, P-A., Appel, R. D., et al. (2001) The mouse SWISS-2D PAGE database: a tool for proteomics of diabetes and obesity. Proteomics 1, 136–163
• Ünlü, M., Morgan, M. E., and Minden, J. S. (1997) Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077
• Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J., et al. (2001) Validation and development of fluorescence two-dimensional differential gel electrophoresis proteomics technology. Proteomics 1, 377–396
• Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., Flaig, M., et al. (2002) 2-D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers. Mol. Cell. Proteom. 1, 117–123
• Gharbi, S., Gaffney, P., Yang, A., Zvelebil, M. J.,Cramer, R., Waterfield, M. D., and Timms, J. F.(2002) Evaluation of two-dimensional differential gel electrophoresis for protein expression analysis of a model breast cancer cell system. Mol. Cell. Proteom. 1, 91–98
• Alban, A., David, S. O., Bjorksten, L., Anderson, C., Sloge, E., Lewis, S., and Currie, I. (2003) A novel experimental design for comparative two-dimensional gel analysis: two-dimensional gel electrophoresis incorporating a pooled internal standard. Proteomics 3, 36–44
• Hanlon, W. A., and Griffith, P. R. (2004) Protein profiling using two-dimensional gel electrophoresis with multiple fluorescent tags. In Proteome Analysis. Interpreting the Genome (Speicher, D. W., ed.), Elsevier, Amsterdam, pp. 19–73
• Link, A. J. (1999) Autoradiography of 2D gels. Methods Mol. Biol. 112, 285–290
• Patterson, S. D., and Latter, G. I. (1993) Evaluation of storage phosphor imaging for quantitative analysis of 2D gels using the Quat II system. Biotechniques 15, 1076–1083
• Monribot-Espagne, C., and Boucherie, H. (2002) Differential gel exposure, a new methodology for the two-dimensional comparison of protein samples. Proteomics 2, 229–240
• Görg, A., Obermaier, C., Boguth, G., and Weiss, W. (1999) Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis 20, 712–717
• Santoni, V., Rabilloud, T., Doumas, P., Rouqui, D., Mansion, M., Kieffers, S., et al. (1999) Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels. Electrophoresis 20, 705–711
• Schüpbach, J., Ammann, R. W., and Freiburghaus, A. U. (1991). A universal method for two-dimensional polyacrylamide gel electrophoresis of membrane proteins using isoelectric focusing on immobilized pH gradients in the first dimension. Anal. Biochem. 196, 337–343
• Rabilloud, T. (1998) The use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19, 758–760
• Chevallet, M., Santoni, V., Poinas, A., Rouquie, D., Fuchs, A., Kieffer, S., et al. (1998) New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis. Electrophoresis 19, 1901–1090
• Maserti, B. E., Della Croce, C. M., Luro, F., Morillon, R., Cini, M., and Caltavuturo L. (2007) A general method for the extraction of citrus leaf proteins and separation by 2D electrophoresis: A follow up. J. Chromatogr. B 849, 351–356
• Fountoulakis, M., Juranville, J. F., Roder, D., Evers, S., Berndt, P., and Langen, H. (1998) Reference map of the low molecular mass proteins of Haemophilus influenzae . Electrophoresis19, 1819–1827
• Fountoulakis, M., Berndt, P., Langen, H., and Suter, L. (2007) The rat liver mitochondria proteins. Electrophoresis. 23, 311–328
• Molloy, M. P., Herbert, B. R., Walsh, B. J., Tyler, M. I., Traini, M., Sanchez, J-C., et al. (1998) Extraction of membrane proteins by differential solubilisation for separation using two-dimensional electrophoresis. Electrophoresis19, 837–844
• Nouwens, A. S., Cordwell, S. J., Larsen. M. R.,Molloy, M. P., Gillings, M., Willcox, M. D., and Walsh, B. J. (2000) Complementary genomics with proteomics: the membrane bproteome of Pseudomonas aeroginosa PAO 1. Electrophoresis 21, 3797–3809
• Molloy, M. P., Herbert, B. R., Williams, K. L.,and Gooley, A. A. (1999) Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis. Electrophoresis 20, 701–704
• Ferro, M., Seigneurin-Berny, D., Rolland, N., Chapel, A., Salvi, D., Garin, J., and Joyard, J. (2000) Organic solvent extraction as a versatile procedure to identify hydrophobic chloroplast membrane proteins. Electrophoresis 21, 3517–3526
• Fujiki, Y., Hubbard, A. L., Fowler, S., and Lazarow, P. B. (1982) Isolation of intracellular preparations by means of sodium carbonate treatment: applications to endoplasmic reticulum. J. Cell Biol. 93, 97–102
• Bjellqvist, B., Sanchez, J. C., Pasquali, C., Ravier, F., Pasquet, N., Frutiger, S., et al. (1993) A nonlinear wide-range immobilised pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14, 1357–1365
• Poland, J., Cahill, M. A., and Sinha, P. (2003) Isoelectric focussing in long immobilized pH gradient gels to improve protein separation and proteome analysis. Electrophoresis 24, 1271–1275
• Hoving, S., Voshol, H., and van Oostrum, J.(2000) Towards high performance two-dimensional electrophoresis using ultrazoom gels. Electrophoresis 21, 2617–2621
• Sabounchi-Schutt, F., Astrom, J., Eklund, A.,Grunewald, J., and Bellqvist, B. (2001) Detection and identification of human bronchoalveolar lavage proteins using narrow-range immobilized pH gradient Drystrips and paper bridge sample application method. Electrophoresis 22, 1851–1860
• Westbrook, J. A., Yan, J. X., Wait, R., Welson, S. Y.,and Dunn, M. J. (2001) Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms. Electrophoresis 22, 2865–2871
• Görg, A., Boguth, G., Köpf, A., Reil, G., Parlar, H., and Weiss, W. (2002) Sample preparation with Sephadex isoelectric focusing prior to narrow pH range two-dimensional gels. Proteomics 2, 1652–1657

Advertisement

Electrophoresis: Principles, Types, and Uses

Electrophoresis is a simple and sensitive separation technique in clinical and research laboratories. Since its discovery, it has been an essential tool used by biologists and chemists to separate mixtures, especially proteins and nucleic acids.

Electrophoresis consists of two words; electro , meaning electricity, and phoresis, meaning movement. Thus, it implies the migration and separation of a charged particle (ions) through a solution under the influence of an electric field.

It was first demonstrated in 1807 by Ruess, who noted the migration of particles towards the anode. It has improved from the initial crude paper electrophoresis to the modern automated system. Various improved versions are available, which apply in miniaturization, precision engineering, etc. The benefit of advancement is meeting the requirement for faster and better resolution of results.

Table of Contents

Principle of Electrophoresis

Biological molecules, like amino acids, peptides, proteins, nucleic acids, and nucleotides, possess ionizable groups. These molecules exist in solution as electrically charged species, cations (+), or anions (-) at any given pH. Thus, the electric field allows the migration of the negatively charged molecule towards the anode (a positive terminal). In contrast, the positively charged molecule migrates towards the cathode (a negative terminal).

The separation of the molecules, ions, or colloidal particles suspended in the matrix occurs due to the force of an electric field. The molecules move through a sieve-like compound based on the molecular mass and charge ratio.

It is an incomplete form of electrolysis as the electric field is removed before the molecules reach the electrode, yet the molecules separate due to electrophoretic mobilities. 

Nucleic acids have negative phosphate backbones. Hence they move towards the anode in DNA electrophoresis. Ampholytes, like proteins, bear both positive and negative charges. Such compounds have negative charge in normal conditions and migrate towards the anode. At the same time, they are positively charged in acidic conditions and move towards the cathode. Hence, protein bears a negative or positive charge depending on solvent pH and isoelectric point.

Factors Affecting the Rate of Ion Mobility

The velocity of ions depends on both inherent factors and the external environment.

Inherent factors

The inherent factors that affect the velocity of ions are:

• Charge density
• Molecular weight
• The net charge of the molecule
• Size and shape of the molecule

External factors

The external factors affecting the rate of movement of ions are:

• Electrical parameters, like current, voltage, and power
• Viscosity and pore size of supporting medium
• Temperature
• The pH of the buffer

Electrophoresis Instrument

Modern electrophoresis equipment and systems vary based on its types and forms. However, all the electrophoretic system possesses two essential components:

Power supply drives the movement of ionic species in the medium and allows adjustment and control of either the current or the voltage.

• An electrophoresis unit

An electrophoretic system depends on its type but essentially consists of two electrodes of opposite charge (anode and cathode), connected by a conducting medium called an electrolyte. In addition, a supportive medium is present in electrophoretic systems like gel and paper electrophoresis.

• Buffer (Electrolyte)

Buffers carry applied electric current and provide appropriate pH for the process. Conducting (running) buffers like Tris borate EDTA (TBE) and Tris-acetate acid EDTA (TAE) are commonly used.

• Supportive Medium

The supportive medium is the matrix (gel), in which biomolecules are separated. It can be in the slab or capillary form. The supportive mediums used are sugar polymers like agarose gel, polyacrylamide gel, starch gel, and cellulose acetate gel. The medium runs either vertical or horizontal gel systems in gel electrophoresis. Horizontal: agarose gel electrophoresis, and vertical: SDS-PAGE. The higher the pore size, the higher the speed traveled by charged particles.

General Procedure of Electrophoresis

The electrophoresis process has three main steps; separation, detection, and quantification.

The instrument set up is according to its type. In the gel electrophoresis, gels are prepared and cast. Then placed into the electrophoresis chamber. The supportive medium can be agarose gels or polyacrylamide gels. Then appropriate buffer solution is added to the system.

After the proper setup of the instrument, the sample is placed into the medium. Then the sample is run at a specific current, voltage, or power.

Detection and Quantification

Staining with a dye or autoradiography (for radioactive samples) helps in the detection of the separated components.

Quantification is done using a densitometer or by direct measurement using an optical detection system. For example, protein is fixed by precipitating in gel with acetic acid. Methanol helps prevent the diffusion of proteins from the gel during the staining process.

Forms of Electrophoresis

Based on the forms, it is of two types; zone and moving boundary electrophoresis.

In the moving boundary electrophoresis , charged molecules migrate in a free-moving solution without a supporting medium. E.g., Capillary electrophoresis.

Types of Electrophoresis

Based on the nature of the supporting medium, it is of the following types:

• Agarose gel electrophoresis
• Polyacrylamide gel electrophoresis
• Cellulose Acetate Electrophoresis
• Capillary Electrophoresis

Depending on the mode of technique, it has the following types:

• Paper electrophoresis
• Isoelectric focusing electrophoresis
• Two-dimensional Polyacrylamide gel electrophoresis
• Pulse field gel electrophoresis
• Immunoelectrophoresis
• Capillary electrophoresis
• High voltage electrophoresis
• Isotachophoresis
• Microchip electrophoresis

Uses of Electrophoresis

It is applied for routine laboratory experiments, disease diagnosis, research-oriented separations and identification. Similarly, it is used in various other fields, like forensics, agriculture, pharmaceutical, foods, etc. Some of its applications are described below:

DNA Analysis and DNA Fragmentation

Gel electrophoresis is the core technique for genetic analysis and purification of nucleic acids for further studies or disease diagnosis.

Identifying Specific protein

• The rate of movement of macromolecules in an electric field is a helpful parameter to know any changes in amino acids regarding their charge.
• Quantitative analysis of specific serum protein classes such as gamma globulins and albumins
• It helps in the identification and quantitation of hemoglobin and its subclasses.
• It also helps in the identification of monoclonal protein in either serum or urine.
• Likewise, it helps in the separation and quantitation of significant lipoprotein classes.
• Immunoelectrophoresis helps to analyze several kinds of protein’s existence and how they behave chemically in different environments.
• It is also helpful in purifying proteins for different purposes.
• Similarly, it is useful in determining the molecular weights of protein.

Coenzymes separation

It is useful in separating and quantifying coenzymes such as creatine, kinase, lactate dehydrogenase, and alkaline phosphatase coenzyme to their respective subtypes.

Analysis of chemical compounds

• It helps analyze compounds, such as water, soil, air quality or contamination, food quality, processing hygiene, and medical forensic analysis.
• It also helps in analyzing transition metals.
• Likewise, it helps to analyze organic compounds.
• Similarly, it helps in analyzing components of pesticides.
• Angerish A (2018). Study of Electrophoresis Techniques and its Types. Journal of Emerging Technologies and Innovative Research. 5(9): 299-304.
• Fritsch, R., & Krause, I. (2003). ELECTROPHORESIS. Encyclopedia Of Food Sciences And Nutrition , 2055-2062. https://doi.org/10.1016/b0-12-227055-x/01409-7
• Chin et al. (2013). Electrophoresis: What does a Century-Old Technology Hold for the Future of Separation Science? International Research Journal of Applied and Basic Sciences. 7(4): 213-221.
• Westermeier, R. (2005). Gel Electrophoresis. Els . https://doi.org/10.1038/npg.els.0005335
• Wilson K & Walker J (1994). Principles and Techniques of Practical Biochemistry: Electrophoretic Techniques. 4 th Ed. Cambridge University Press. 4 th Ed: 425-460.

Srijana Khanal

Hello, I am Srijana Khanal. Former faculty teacher in Microbiology Department at National College, NIST. Involved in the field of teaching for almost 10 years. I am very passionate about writing (academic as well as creative). My areas of interest are basic science, immunology, genetics, and research methodology.

Recent Posts

Pulsed-Field Gel Electrophoresis (PFGE): Steps, Applications

Pulsed-field gel electrophoresis (PFGE) is a powerful molecular typing technique by which genomic DNA is isolated from the organism of interest, followed by restriction enzyme analysis. The digestion...

Agarose Gel Electrophoresis: Principle, Procedure, Results

Agarose gel electrophoresis is one of the most common electrophoresis techniques which is relatively simple and straightforward to perform but possesses great resolving power. The agarose gel...