Consider a slab gel – polyacrylamide that is impregnated with “ampholytes”
Ampholytes are a mixture of synthetic polymers (usually amino acids) that vary in their IEP, say from pH 3 to pH 10. The ampholytes are usually pre-focused (but don’t have to be) to set up a pH gradient in the gel
The samples for analysis are NOT treated with SDS or reductant – they are added to the IEF gel in their native state with a small amount of ampholytes
Samples are added to the Gel and gel is placed in apparatus. The upper chamber is filled with anolyte (10 mM phosphoric acid) and the lower chamber is filled with catholyte (20 mM NaOH). The electric field is then turned on.
The ampholytes and sample proteins will migrate until their charge is neutral, then they don’t migrate any more. Thus a stable pH gradient is set up, and molecules are separated based on their differences in IEP