1. Cause in normal IEF CBB staining:
During the fixing process with 20% TCA the Ampholytes who are producing the pH-gradient should leave the gel completely. If certain Ampholytes will not do this then they get stained like the proteins because they are also Amino-carbon-acids.
If alcohol-containing staining recipes are used, the staining and the destaining solution should be more lipophylic.
Slightly increase the alcohol compound of the washing solution. See table below.
The increasing should be done very carefully because lipophylic proteins get soluble too and will be not visible anymore after the visualization!
If Serva-Violet staining is used no other Ampholytes should be inside the gel.