Western Blot - 2-D Gel Electrophoresis

2-D Gel Electrophoresis

2-dimensional SDS-PAGE uses the principles and techniques outlined above. 2-D SDS-PAGE, as the name suggests, involves the migration of polypeptides in 2 dimensions. For example, in the first dimension polypeptides are separated according to isoelectric point, while in the second dimension polypeptides are separated according to their molecular weight. The isoelectric point of a given protein is determined by the relative number of positively (e.g. lysine and arginine) and negatively (e.g. glutamate and aspartate) charged amino acids, with negatively charged amino acids contributing to a high isoelectric point and positively charged amino acids contributing to a low isoelectric point. Samples could also be separated first under nonreducing conditions using SDS-PAGE and under reducing conditions in the second dimension, which breaks apart disulfide bonds that hold subunits together. SDS-PAGE might also be coupled with urea-PAGE for a 2-dimensional gel.

In principle, this method allows for the separation of all cellular proteins on a single large gel. A major advantage of this method is that it often distinguishes between different isoforms of a particular protein - e.g. a protein that has been phosphorylated (by addition of a negatively charged group). Proteins that have been separated can be cut out of the gel and then analysed by mass spectrometry, which identifies the protein.

Please refer to reference articles for examples of the application of 2-D SDS PAGE.