Question:
What are the techniques used for protein electrophoresis?
The Protein Man Says:
Protein electrophoresis is a commonly used technique in proteomics that aims to separate individual protein components in a sample based on their physical properties. This technique facilitates the separation of different protein types by passing an electrical current through a polyacrylamide sieving gel matrix. As can be expected, the charged protein particles will migrate through the matrix allowing for an accurate detection of individual protein components present in the sample.
There are a number of techniques by which protein components can be separated and detected using electrophoresis. This includes native or non-denaturing PAGE, SDS-PAGE, tris tricine, bis tris, isoelectric focusing (IEF) and zymography.
Protein Electrophoresis Techniques and Applications
Native PAGE. This technique was developed by Ornstein and Davis in 1964 to separate proteins based on their mass and native charge while preserving their native protein conformation and biological activity. While this technique effectively separates protein components in their native state, it often yields unpredictable separation patterns that may affect the result of your research.
SDS-PAGE. The SDS-PAGE is the most commonly used technique in separating protein components based on mass. This technique was developed by Professor Ulrich Laemlli in 1970 to overcome the apparent limitations of the native PAGE technique. It uses sodium dodecyl sulphate (SDS) to denature the protein components in the sample. The SDS also functions to form a non-covalent bond with the protein components to impart an overall negative charge and change its complex tertiary shape to create a long, rod-like conformation. This allows the proteins to move through the gel matrix depending on its size and allows for easier molecular weight determination.
Tris Tricine. This electrophoresis technique is most commonly employed in the separation of small proteins and peptides with a molecular weight of less than 10 kDa. Available buffers include tricine sample buffer, tris-tricine and tris-tricine-SDS.
Bis Tris. You should use this technique if you are separating small to medium sized proteins under denaturing conditions. In this particular technique, different separation ranges can be achieved by using MES SDS or MOPS/SDS running buffers.
Isoelectric Focusing (IEF). IEF is best used in separating proteins based on the differences in their isoelectric points. While this technique can very well be used independently, it is commonly used as the first dimension of 2D PAGE.
Zymography. This technique is primarily based on SDS-PAGE and includes the use of an enzyme substrate co-polymerized with the polyacrylamide gel to facilitate accurate detection of enzyme activity. In this technique, samples are prepared in Zymogram sample buffer without boiling or using any reducing buffer to retain the native structure and activity of the enzyme. The SDS is then washed with Zymogram Renature Buffer following electrophoresis, incubated in an appropriate developing buffer and stained with Amido Black or Coomassie Brilliant Blue to produce clear bands against a darkly stained background. These bands represent the areas of digestion.
