Electrophoresis is a powerful technique for finer protein separation and visualization of separated proteins. It is based on the principle of migration of charged proteins in an electric field. Electrophoretic techniques have developed significantly for improved protein separation and detection. Learning Objective After interacting with this Learning Object, the learner will be able to: Utilize the knowledge of Two Dimensional Electrophoresis (2-DE). Illustrate the concept of difference Gel Electrophoresis (DIGE).
2-DE, Isoelectric focusing Prior to isoelectric focusing in 2-DE, the commercially available IPG strips must be rehydrated.
2-DE, Isoelectric focusing This can be done either by passive rehydration or active rehydration. In passive rehydration, the IPG strip is placed with its gel-side downwards in a well containing the protein sample reconstituted with a suitable buffer solution. This is then covered with mineral oil to prevent the gel from drying up and left overnight for 10-20 hours depending on the length of the strip.
2-DE, Isoelectric focusing In active rehydration, the protein sample is added to the strip via a sample cup followed by cover fluid to prevent the gel from drying up. This is then placed in the isoelectric focusing instrument and low voltage is applied, which allows the strip to take up the protein sample. Active rehydration is also performed for 10-20 hours depending upon the length of IPG strip being used.
2-DE, SDS-PAGE These loaded strips are then focused on an isoelectric focusing unit by passing current. The various proteins of the sample mixture migrate in the electric field and come to rest when the ph is equal to their pi i.e. they become neutral and are no longer affected by the electric field. Progress of electrophoresis is monitored by means of a tracking dye like bromophenol blue (BPB) which is a small molecule and therefore migrates ahead of all other proteins.
2-DE, SDS-PAGE The IPG strip is equilibrated in a reducing agent like DTT followed by an alkylating agent, iodoacetamide which prevents reformation of the reduced bonds. This strip containing the separated proteins is then placed on the SDS-polyacrylamide gel slab for further protein separation in the second dimension based on their molecular weight.
2-DE, SDS-PAGE The proteins on the IPG strip are then subjected to SDS-PAGE by applying a direct current between 100-350V depending upon the size of the gel. Any proteins that may have been present as a single band on the IPG strip due to similar isoelectric points can now be separated on the basis of their molecular weight with smaller proteins migrating farthest.
2-DE, SDS-PAGE View of a sample gel which has been run by 2-DE and stained with Coomassie blue. Each spot provides information about the MW and pi of the proteins.
Internal standard preparation The pooled internal standard for DIGE is prepared by mixing equal amounts of all samples that are being run in the experiment. This prevents problems of gel-togel variations.
Dye labeling Each protein sample as well as the internal standard is labeled with a differently fluorescing cyanine dye which allows all protein samples to be simultaneously run on a single gel. The dye binds covalently to the εamino group of lysine residues in proteins.
Mixing and 2-DE The labelled protein samples are mixed and run on a single 2-DE gel. Separation takes place on the basis of isoelectric points of the proteins in one dimension and based on molecular weight of the proteins in the second dimension with the smaller proteins migrating further along the gel.
Gel viewing The gel containing all the protein samples can be viewed by illuminating it alternately with excitation wavelengths corresponding to the various Cyanine dyes.
Gel viewing View of a superimposed DIGE gel depicting all protein spots of multiple samples. Information on molecular weight and pi of proteins can be obtained from these spots.
Protein separation Techniques