Protein Detection

摘要

This chapter describes protein detection techniques used in gel electrophoresis. Gel-based methods allow both prelabeling and poststaining. After electrophoretic separation, the protein zones have to be prevented from diffusion and elution from the gel matrix. The fixation step is particularly critical after isoelectric focusing (IEF) because the proteins need to be fixed in the matrix while the carrier ampholytes must be washed out to avoid background staining. In earlier protocols, fixation recipes using different mixtures of trichloroacetic acid (TCA), sulfosalicylic acid and perchloric acid can be found for fixing the proteins after IEF. The first dye used for protein detection in gels was Amido Black 10 B, which has been known from clinical serum electrophoresis in cellulose acetate membranes and agarose gels. For difference gel electrophoresis (DIGE), the sample proteins are labeled with modified fluorescent cyanine dyes. The chapter also discusses image analysis, manual and robotic spot-picking and semiautomated picking.