First dimension electrophoresis (isoelectric focusing) is performed under denaturing conditions which gives the highest resolution and the cleanest results.  The IEF is the most critical step of the 2-D electrophoresis process as proteins need to be solubilized without charged detergents like SDS (it can give extra charge on protein and changes isoelectric point). Most common protocol uses high concentration of urea and reducing agents. First dimension separation also requires selecting a 1st dimension pH range (depends on sample) and length of strip (depends on size of second dimension gel). After first dimension run, strip gel is placed on the top of SDS-PAGE gel horizontally (a normal SDS-discontinuous system is used). The protein is already denatured in first dimension (by urea and reducing agents). Thus, the SDS used in gel running buffers sufficient to bind with already denatured protein maintain the necessary uniform negative charge for SDS-PAGE.

[Recall procedure in normal SDS-PAGE we studied in previous lecture: The process involves boiling of sample with reducing agent and SDS so that protein can denature and bind with SDS to give uniform negative charge). Two dimensions of t wo-dimensional gel electrophoresis (2-D electrophoresis) is shown in Fig. 1

Figure 1: Two dimensions of t wo-dimensional gel electrophoresis (2-D electrophoresis)

Once the second dimension run is over, gel is separated and stained for protein visualization using methods studied in SDS-PAGE (Coomassie blue staining, Silver staining etc.)