|TThe proteins separated in IPG strips by IEF in the first dimension are applied to polyacrylamide gels and separated a second time by SDS-PAGE (Figure bellow) by size (molecular weight) in a direction perpendicular to the first dimension.||
The second-dimension separation is by protein size (mass) using SDS-PAGE. Vertical electrophoresis cells are plastic boxes with anode and cathode buffer compartments that contain electrodes. The electrodes (typically platinum wire) connect to a jack attached to a power supply. The gels are held vertically between the electrode chambers during electrophoresis (Andrews 1986)1. Vertical electrophoresis cells are made in different size formats to accommodate different gels sizes. Deciding which cell to use depends on the requirements for speed, resolution, and throughput (both the number of samples and gels) as well as the volume of sample available. The proteins separated in IPG strips by IEF in the first dimension are applied to polyacrylamide gels and separated a second time by SDS-PAGE by size (molecular weight) in a direction perpendicular to the first dimension.
Prior to second-dimension separation, an equilibration step is applied to the IPG strip containing the separated proteins. This process reduces any disulfide bonds that may have re-formed during the first dimension and alkylates the resultant sulfhydryl groups.
High-resolution 2-D methods enable separation of thousands of polypeptides in a single slab gel. The resulting spots can be visualized by gel staining, or they can be transferred to a membrane support for total protein staining or analysis with specific antibody detection.
The proteins are complexed with SDS, reduced with DTT, and then alkylated with iodoacetamide. Treatment of the proteins with SDS yields protein-SDS complexes with a consistent charge-to-mass ratio. When electrophoresed through a polyacrylamide gel, these complexes migrate with a mobility that is related logarithmically to mass. As the proteins migrate through the gel, the pores of the gel sieve proteins according to size.
1. Andrews AT (1986). Electrophoresis: theory, techniques and biochemical and clinical applications (New York: Oxford University Press).