Electrophoresis is a method used to separate macromolecules based on their charge, binding affinity, and size under an electric field. It is broadly categorized into capillary type and slab type electrophoresis. Polyacrylamide gel electrophoresis (PAGE) is a type of capillary type electrophoresis.
Polyacrylamide gel electrophoresis is a subtype of gel electrophoresis applicable in molecular biology, forensic chemistry, genetics, biochemistry, and biotechnology to separate biological macromolecules, primarily proteins or nucleic acids, based on their electrophoretic mobility. This technique replaces the regular gel with polyacrylamide gel as a support matrix. The most commonly used polyacrylamide gel electrophoresis for quantitative protein analysis is Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
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Polyacrylamide Gel
The polyacrylamide gel forms by polymerizing acrylamide and a crosslinking agent, i.e., N, N’-methylene-bis-acrylamide. It does not react with proteins and consists of pores and channels that allow the protein to move through it. Two parameters characterizes polyacrylamide gel: total monomer concentration (% T, in g/100 ml) and weight percentage of crosslinker (%C). Deferring these two parameters, helps in regulation in the pore size of the gel to yield the best separation result. %T indicates the pore size of the prepared gel.
Principle of Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis is based on the principle that charged particles migrate to the electrode of the opposite sign under the influence of an electric field.
In polyacrylamide gel electrophoresis, the sample (i.e., protein) dissolves into a buffer containing denaturing agents (i.e., sodium dodecyl sulfate, and β-mercaptoethanol ), glycerol, bromothymol blue. An anionic detergent, sodium dodecyl sulfate, helps denature the sample protein and binds to the side chain of amino acids at one SDS anion per two residues.
Therefore, the negative charge of SDS results in a net negative charge in the protein sample. Similarly, β-mercaptoethanol also denatures the protein sample by cleaving the disulfide bond. Furthermore, glycerol helps to increase the density of the protein sample so that the protein sample falls at the bottom of the gel rather than floating or mixing with the running buffer. Likewise, bromothymol blue helps to visualize the protein sample in a gel during electrophoresis.
When the electric current passes through the electrophoretic chamber, the protein-sodium dodecyl sulfate complex starts moving toward the anode. The gel with a larger pore size (containing 4-8% acrylamide) permits higher molecular weight molecules to move faster through the gel, while the gel with a smaller pore size (containing 12-20% acrylamide) restricts the migration of larger molecules allowing only passage of smaller molecules.
Materials Required for PAGE
Various materials are required to conduct polyacrylamide gel electrophoresis, which include;
- Polyacrylamide gel: It is the matrix that helps to separate proteins based on their size. It can be either prepared in the lab or can be purchased.
- Running buffer: It varies based on sample type. Its primary function is to allow the conduction of current across the gel.
- Protein or nucleic acid sample: The primary sample needed to separate based on its molecular weight.
- Staining and de-staining reagent: Coomassie stain solution is mainly helps to stain protein bands after electrophoresis. In contrast, the de-staining reagent (prepared by mixing methanol, acetic acid, and water) helps to de-stain the gel.
Essentials of electrophoresis:
- Gel plate: It is the plate that holds the polymerized gel in an electrophoresis chamber.
- Comb: It helps to create a well (place for loading primary sample) in a separating gel.
- Electrophoresis chamber: Polyacrylamide gel is packed within a running buffer, and electrophoresis is carried out.
- Protein ladder: It is a reference protein ladder with known size. It helps to confirm the molecular weight of the protein of interest.
- Power supply: It supports converting AC to DC, which is essential to create an electric field.
Procedure involved in PAGE
The procedure involved while operating polyacrylamide gel electrophoresis is;
Sample preparation
- The sample can be either protein or nucleic acid.
- The mixing of sample occurs in a loading buffer containing denaturing agents (i.e; SDS, and β-mercaptoethanol), glycerol, and bromothymol blue. Unlike protein, urea can help as a denaturing agent for nucleic acid.
- Heating the samples with denaturing agents and mercaptoethanol for 5-10 minutes further enhances the denaturation.
Preparation of polyacrylamide gel
- It comprises acrylamide, bisacrylamide, optional denaturant (SDS for protein and Urea for nucleic acid), and buffer with adjusted pH.
- All of the reagents are combined except TMED. TMED is mixed when the gel is ready to pour. Similarly, butanol is also poured into it to avoid bubble formation.
- Two types of gels, i.e., stacking gel pH 6.8 and separating gel pH 8.8, are prepared. Furthermore, at first, separating gel is poured, and over it stacking gel is run, ensuring all of the sample proteins arrive at the separating gel simultaneously.
- After polymerization, the comb is inserted within the stacking gel layer on a glass plate.
- The polymerized gel is then known as a ‘gel cassette.’
Electrophoresis
- Depending on the sample type, the use of different buffer systems can help to run polyacrylamide gel electrophoresis.
- The buffer used at the cathode or anode might be the same or different.
- During electrophoresis, the gel cassette was removed from the casting stand and was placed in the electrode assembly. It is later on fixed in the clamp stand.
- Then, 1X running buffer is poured into the electrophoresis chamber.
- Each well is then loaded with a protein sample. Similarly, marker protein is also loaded into a single well of gel.
- The tank is then covered with a lid, and the sample can run at 30mA for about 1 hour.
Detection
- Following electrophoresis, the gel may be stained with a staining reagent (coomassie for proteins or ethidium bromide for nucleic acids). Staining helps to visualize distinct bands that can be compared with marker bands.
Applications of Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis is applicable in a wide range of that includes;
- It helps to determine the purity of samples.
- It is also helpful in determining the molecular weight of protein.
- PAGE helps to quantify the proteins.
- It helps in monitoring changes in protein in body fluids.
- PAGE is useful in peptide mapping.
- It is useful to estimate the purity of the protein and nucleic acid.
- PAGE is helpful to perform western blotting after electrophoresis.
- It is useful for the detection of protein ubiquitination.
- PAGE is helpful to analyze the size and number of polypeptide subunits.
- It is useful in HIV tests to separate HIV protein.
Advantages of Polyacrylamide Gel Electrophoresis (PAGE)
Polyacrylamide gel electrophoresis consists of the following benefits:
- It helps to determine the molecular weight, as migration is directly proportional to the molecular weight.
- It is a susceptible test.
- It can provide results even with a small amount of sample.
- It consists of chemically crosslinked stable gel.
- The sample recovered from the gel is exceptionally pure.
- It is best for separating proteins of low molecular weight.
Disadvantages of Polyacrylamide Gel Electrophoresis (PAGE)
Despite the benefits, polyacrylamide gel electrophoresis has some drawbacks which are as follows:
- Chemical acylamide is a potent neurotoxin.
- Preparation of gel is time-consuming, and electrophoresis also takes a longer time.
- It requires a higher budget to operate.
- The preparation of a new gel for each test is necessary.
Precautions of Polyacrylamide Gel Electrophoresis (PAGE)
While performing polyacrylamide gel electrophoresis one should consider following precautionary measure:
- Routine care should be exercised in handling buffers and samples to avoid accidental ingestion, needle stick, etc.
- Acrylamide and bisacrylamide are highly toxic monomers of polyacrylamide gel. Therefore, the use of gloves for handling polyacrylamide gel.
- A precise amount of polyacrylamide monomers should be weighed to obtain desired pore size.
- Always wear gloves, goggles, and a lab coat while handling samples and buffers.
Differences Between Agarose Gel and Polyacrylamide Gel Electrophoresis (PAGE)
Although both techniques are used for separation purposes, they have some differences. The differences between agarose gel electrophoresis and polyacrylamide gel electrophoresis are given below;
| Polyacrylamide gel electrophoresis (PAGE) | Agarose gel electrophoresis |
| In polyacrylamide gel electrophoresis, polyacrylamide gel separates macromolecules, i.e., proteins of size five kDa to 250 kDa. Similarly, it can also isolate DNA of 5- 500 bp size. | In agarose gel electrophoresis, agarose gel separates DNA, RNA, and protein. It can isolate DNA about 50-20,000 bp in size. |
| The run configuration of polyacrylamide gel electrophoresis is vertical. | The run configuration of agarose gel electrophoresis is horizontal. |
| Polyacrylamide powder or gel is toxic. | Agarose gel is considered entirely non-toxic. |
| It gives better resolution than agarose gel. | It gives poor resolution than polyacrylamide gel. |
| PAGE can not be reused. | Agarose gel electrophoresis can be reused if necessary. |
| It requires two layers of gel, i.e., staking and separating gel. | It only requires a single layer of agarose gel. |
References
- Walker, J. M. (2010). 10 Electrophoretic techniques. In K. Wilson & J. M. Walker (Eds.), Principles and Techniques of Biochemistry and Molecular Biology (7th ed.). Cambridge: Cambridge University Press.
- Robin M. Zuck. SOP: SDS-PAGE Protein Gel Electrophoresis. Retrieved on 12th march 2023. Retrieved from https://biomanufacturing.org/uploads/files/59833385998409326-qcb-15-sds-page-protein-gel-electrophoresis.pdf
