Critical: A) Visualizations with many steps like silver and fluorescence and/or alcohol-containing recipes. Peptides will leave the gel before you will see them. As visualisation we suggest to do the “Hot Coomassie Staining”, the “Semi Colloidal Coomassie” or the “Blakesley” (Glycopeptides) B) SDS-Electrophoresis: The peptides appear there, where the SDS-cloud is located (before the anode). SDS will not dock on smaller and / or basic peptides --> Anodal Native - Cathodal Native As second dimension of a 2-dimensional electrophoresis it also can be critical: Beside classical 2D-methods for peptides (SDS-Peptides-2D): Double Native 2D, or Double Dimension 2D C) IPG-focusing in the basic region: Some basic peptides will not work! --> basic peptides
The 2-dimensional possibilities:
To see all peptides / proteins in 1 separation the classical method should be used: First dimension = IEF, second dimension = SDS-electrophoresis
To work without SDS means that 2 different native electrophoresis’ have to be performed: Double Native Electrophoresis for acidic and for basic peptides First dimension = IEF, second dimension = native electrophoresis The 2 native electrophoresis’ show good transfer from the IPG-strips and are easy to perform.
If basic peptides / proteins give no good results in isoelectric focusing: Double Dimensional Electrophoresis First dimension = native electrophoresis, second dimension = SDS-electrophoresis