Protein gel electrophoresis, or simply protein electrophoresis, is a common technique used in laboratories to separate biomolecules, including DNA, RNA, and proteins, based on size or molecular weight.
Gel electrophoresis of proteins is a standard laboratory technique in which charged proteins are transported through a gel matrix by passing an electric field through a solvent. It’s a simple, sensitive, and rapid analytical tool.
The two support matrices used in electrophoresis are polyacrylamide and agarose. They serve as a molecular sieve through which biomolecules are separated based on their molecular mass. Though agarose has a large pore size, it’s used to separate large protein complexes and nucleic acids. Polyacrylamide, in contrast, has small pore sizes. It’s suitable for separating smaller proteins (5-250 KDa) and small nucleic acids of 5-500 base pairs.
To track the movement of proteins in the gel matrix during electrophoresis, a tracking dye is used in the sample buffer. A very common dye is Bromophenol Blue, which is colored at neutral and alkali pH. The dye contains a negative charge, moving it towards the anode along with the proteins during electrophoresis.
Afterwards, fixed and separated protein bands on the gel matrix are visualized using gel staining. A common protein stain used in labs is Coomassie Brilliant Blue R-250. It’s also used during the Bradford assay. The dye strongly binds to the proteins in the gel, giving them a deep blue color and leaving the acrylamide colorless, making it simple to visualize fixed and separated bands. The silver staining procedure can also be used for gel staining. It can detect even trace amounts of proteins in gels, nucleic acids, or polysaccharides.
Protein gel electrophoresis is used in labs to analyze protein samples for:
In this article, we will cover more about protein gel electrophoresis, including the various techniques, working mechanisms, and applications in the laboratory.
Polyacrylamide Gel Electrophoresis (PAGE) is a technique used in life sciences and biotech labs to separate proteins and nucleic acids based on their electrophoretic mobility.
Polyacrylamide is a chemical substance used to prepare electrophoretic gels that act as a sieve for biomolecules during electrophoresis. They are prepared by mixing acrylamide with bisacrylamide. The formation of gel occurs as a result of cross-linking and polymerization of these molecules by the addition of a polymerizing agent, ammonium persulfate (APS).
The pores sizes created in the gel are inversely related to the polyacrylamide concentration or percentage. For example, the higher the polyacrylamide concentration, the smaller the pore size. Gels with high percentages are used to resolve small proteins, whereas gels with low percentages are used for large proteins.
Many forms of PAGE are used in labs to extract information on the proteins of interest. Among all, the denaturing sodium dodecyl sulfate PAGE (SDS-PAGE) with a discontinuous buffer system is extensively used in labs to separate proteins.
The SDS-PAGE gel is cast in a buffer containing sodium dodecyl sulfate (SDS), a detergent that denatures protein into polypeptides.
When the protein samples are heated between 70-100°C, in the buffer containing SDS and thiol reagent, it leads to the cleavage of disulfide bonds and the formation of protein subunits. The resulting protein subunits/peptides bind to SDS, which gives them a negative charge and causes them to move towards the anode.
Figure: The effect of SDS on protein charge and conformation.
In labs, SDS-PAGE is one of the most preferred techniques for analyzing proteins because it’s simple and fast, and only requires micrograms of protein samples.
Types of SDS-PAGE based on gel preparation are:
Stacking gel is cast over the top of the resolving gel. It has lower pH (e.g. pH 6.8), lower concentration of acrylamide (e.g., 7% for larger pore size), and different ionic content.
Figure: An illustration of the arrangement of discontinuous gel.
A reference protein with known molecular mass, known as protein ladder, molecular weight marker, or protein size standard, is run alongside the sample proteins in the same gel. It helps in the determination of the mass of the sample proteins.
Figure: A schematic diagram of the complete process of SDS-PAGE.
Native-Page, or non-denaturing PAGE involves separating proteins according to their mass/charge ratio. This technique is used to analyze protein samples in their folded state—no denaturing substance is used.
Proteins gel electrophoresis involves the use of detergent SDS in the process. The presence of SDS (an anionic detergent) in the buffer causes the denaturation of proteins into short peptide chains and provides them with a negative charge.
Therefore, when the electric field is applied the negatively charged ions will start moving towards the anode (positively charged electrode) from the cathode (negatively charged). The smaller proteins or proteins with less molecular mass will move faster compared to proteins with larger mass.
Figure: An illustration of the separation of proteins from the negative to the positive electrode based on their molecular weight.
After separation, the proteins will appear in the form of solid bands that can be visualized using gel stains.
The separated proteins are used for several experimental purposes, including western blotting assay and mass spectrometry analysis. Thus gel electrophoresis is a fundamental step in proteomics studies.
The decision for the use of chemicals for electrophoresis depends on the size and quantity of proteins and the application of the experiment.
For example, Bis-Tris and Tris-glycine are used to separate a broad range of proteins. However, Bis-Tris has greater sensitivity than Tris-glycine in protein detection. It’s suitable to use for applications like mass spectrometry, post-translational modification analysis, or sequencing.
A widely used buffer system is the tris-glycine or “Laemmli” system. It has stacking gel at pH 6.8 and resolving in the range of ~8.3-9.0 pH. However, the limitation of the system is the formation of disulfide bonds between proteins and the inability of reducing agents (present in the loading buffer) to move with the proteins. The challenges can be overcome by using buffers with lower pH, which also provide more stability to the acrylamide gel.
In the majority of labs, researchers cast their gels by following a standard recipe for gel preparation. These gels are known as hand-cast gels.
However, now ready-to-use precast-protein gels are available that can be used for several applications ranging from SDS-PAGE, isoelectric focusing (IEF), to non-denatured protein analysis. The precast gel is more convenient and consistent compared to the hand-cast gels.
Moreover, to optimize protein resolution, shelf life, and run time of gel, they come in different buffer formulations, including Tris-acetate, Tricine Tris-glycine, Bis-Tris. The use of these precast gels also prevents researchers from touching acrylamide, which is a neurotoxin and carcinogen.
Protein gel electrophoresis has a myriad of applications in different life science areas. Some of them include:
In medical labs, protein gel electrophoresis is widely used to analyze blood serum proteins, mainly albumin and globulin. The technique is also useful in the diagnosis of certain diseases, such as multiple myeloma and monoclonal gammopathy.
Additionally, the technique is suitable for the analysis of antibodies, determining the purity and concentration of vaccines, detecting hormones and enzymes in plasma, and determining the right dosage of antibiotics.
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Protein gel electrophoresis is used to separate proteins based on their molecular weight. It can be categorized into different groups based on the protein sample and experimentation goal.
For example, SDS-PAGE is used to perform the complete analysis of proteins by denaturing them into polypeptides and using the separated proteins for further studies, while native-PAGE is used to study proteins without denaturing them, in their native folded state. The buffer systems, including the running buffer and loading buffer of the gel, are also formulated based on the assays’ purposes.
Gel electrophoresis has many applications in medical, clinical, and industrial settings, including studying protein function, regulating protein expression, disease diagnosis, and determining antibiotic dosage. Because of its versatility and ubiquity in basic and translational research, a high-throughput gel electrophoresis system is a must in many different labs.
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