Question:
How 2DE Protein Electrophoresis works?
The Protein Man Says:
Two dimensional protein electrophoresis or 2DE is an established technique commonly used in purifying and analyzing individual proteins from complex biological samples and is currently regarded as the best option for profiling low abundance proteins.By applying this particular technique, the individual proteins are separated from the sample based on their isoelectric points and molecular weights – a process that may seem simple enough in theory but is actually more complex in practice. Here's how it is done.
How Does 2DE Work?
No matter what sample you are working on, 2DE uses four core steps to get the job done – sample solubilization, isoelectric focusing (IEF), SDS-PAGE and protein analysis.
Sample Solubilization
Your samples should be processed and solubilized before loading them into the IEF gel. It is extremely important that you use IEF-compatible lysis reagents (electrically neutral detergents and chaotropes) to maintain the native charge, solubility and relative abundance of your protein of interest. Remember, the success or failure of your experiment may depend on it.
Isoelectric Focusing (IEF)
After solubilizing your sample, you need to load it to your IEF gel. The proteins will be moved through the acrylamide gel by an electric field. Now, since the gel incorporates a pH gradient, the individual proteins will only move until it reaches its isoelectric point (pl) or the pH where the protein has no net charge.
SDS-PAGE
After separating the proteins based on their pl, you need to separate them according to their molecular weights. This can be done by applying an equilibration step to the strip containing the separated proteins (to reduce any disulfide bonds that may have formed during the IEF phase) and treating the proteins with SDS. Once this is done, the proteins will be aligned along two axes – isoelectric point on one end and molecular weight on the other.
Analysis
Depending on the objective of your experiment, you may stain the gels with silver or Coomassie blue to determine total protein, run a western blot on the proteins or excise the proteins from the gel, have them digested and send them out for identification by mass spectrometry (MS).
Two-dimensional protein electrophoresis may require meticulous attention to details and test your patience to its limits but considering the fact that you can use it to separate hundreds or thousands of different proteins simultaneously, you can say that the rewards are well worth the effort.
Image by - L C
