Overview: Electrophoresis Buffers
Gel Electrophoresis is a scientific technique that separates molecules based on a defining physical attribute including size, charge, and shape. This process is used for a variety of purposes in biological sciences, as it allows users to analyze and purify DNA [1], RNA [2], or protein samples [3]. This process can even detect mutations, post-translational modifications, and interactions between target molecules. This procedure typically involves a porous gel produced by mixing and boiling the agarose or polyacrylamide powder into a buffer solution, which eventually results in production of a solidified gel. Molecules of interest can then be mixed with a loading buffer solution, so they can be accurately loaded into wells molded into the gel. Upon carefully loading the samples into their respective wells, an electric current can then be applied to separate/resolve the molecules through the gel based on their size, shape and charge. Smaller molecules often travel much further through the gel than larger ones. By staining the gel with dye, samples can then be made visible.
Image 1: Polyacrylamide gel electrophoresis (PAGE) is one example of gel electrophoresis that can be used to separate protein samples. Not to be confused with agarose gel electrophoresis, PAGE is presented in a vertical gel, where results can be visualized.
There are several different electrophoresis gel options to choose from depending on the desired result. Gels that are made from agarose, a natural polysaccharide with larger pores, are used for larger molecular fragments to resolve through it. Agarose based gel is used in electrophoresis prior to Southern and Northern blotting, where DNA and RNA molecules are transferred onto a membrane and analyzed. Polyacrylamide gel on the other hand is a synthetic polymer with smaller pores and is typically used in electrophoretic separation of proteins prior to Western and Eastern blotting, where proteins are eventually analyzed.
Electrophoresis Buffers
To ensure the proper mechanisms and results are obtained in biochemical experiments such as electrophoresis, the correct buffers must be used. Some important electrophoresis buffers include:
Buffer Type |
Description |
|---|---|
| Gel Casting Buffers | Gel casting buffers are used to prepare the gel for electrophoresis. They contain the gel material, such as agarose or polyacrylamide, and the buffer solution, such as TAE for Agarose gel electrophoresis or Tris-HCl buffer for polyacrylamide gel electrophoresis (PAGE). These buffers provide the ions and the appropriate pH for the separation and are used before any other steps during the electrophoresis process. |
| Sample Buffers | Sample buffers are used to prepare the sample for electrophoresis. These buffers may contain reducing agents such as dithiothreitol (DTT) or Beta-mercaptoethanol that break the disulfide bonds in proteins and a detergent, such as sodium dodecyl sulfate (SDS), denaturing the proteins and rendering them with a net negative charge. They can also be prepared with non-reducing agents, allowing the molecules of interest to move through the gel in different patterns (i.e., under native conditions). Sample buffers also contain a tracking dye, such as bromophenol blue, that allows the monitoring of the migration, and are used before loading the sample into the gel. |
| Gel Running Buffers | Gel running buffers are used to fill the electrophoresis chamber and immerse the gel during the separation. They contain the same buffer solution as the gel casting buffer to keep the conductivity and pH uniform. This buffer is used after loading the sample and sample buffers into the gel wells and before transferring the molecules to the membrane. |
| Transfer Buffers | Transfer buffers are used to transfer the molecules from the gel to the membrane for further analysis such as Northern and Southern blotting for RNA or DNA molecules, respectively, and Western and Eastern blotting for protein molecules and post-translational modification protein molecules. They contain a buffer solution, such as Tris-Glycine (for Proteins) or Tris-Borate (for DNA and RNA), that provide the conductivity and pH. The transfer buffer for proteins requires addition of methanol (10-20% v/v), that enhances the binding of proteins to the membrane. |
| Blocking Buffers | Blocking buffers are used to block the nonspecific binding sites on the membrane after the transfer of proteins from the gel to the membrane, so antibodies bind only to their target protein. The blocking buffers usually contains a protein, such as Bovine Serum Albumin (BSA) or Nonfat Dry Milk, that can coat the membrane an reduce the background noise. This buffer is used before adding the primary antibody in Western blotting or the probe in Southern or Northern blotting. |
| Stripping Buffers | Stripping buffers are used to remove the primary and secondary antibodies from the membrane after the detection of target proteins in Western blotting. It usually contains a low pH solution, such as glycine-HCl, that disrupts the antibody-antigen interactions. This buffer is used after the detection of proteins and before re-probing the membrane with a different antibody. |
Electrophoresis Buffers at Boston BioProducts
Every electrophoresis buffer is unique to the analyte used and the experimental application. Select the appropriate electrophoresis buffer from the catalog or design your optimal formulation with custom manufacturing options at Boston BioProducts.
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References:
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- Aaij, C., & Borst, P. (1972). The gel electrophoresis of DNA. The gel electrophoresis of DNA - ScienceDirect
- Petrov, A., Tsa, A., & Puglisi, J. D. (2013). Analysis of RNA by analytical polyacrylamide gel electrophoresis. In J. N. Abelson, M. I. Simon, & D. W. Moskowitz (Eds.), Methods in enzymology (Vol. 530, pp. 301-313). Analysis of RNA by Analytical Polyacrylamide Gel Electrophoresis - ScienceDirect
- Hames, B. D. (Ed.). (1998). Gel electrophoresis of proteins: A practical approach(3rd ed.).