If your sample contains proteins of interest that are <20 kDa, please download How to Detect Small Peptide Protocol that explains how to detect synthetic peptides using SDS-PAGE, including effective methods for Coomassie blue staining, silver staining, and electroblotting.
Tricine-based SDS-PAGE is used most commonly to separate proteins sized 1–100 kDa and is the electrophoretic system of choice for resolving proteins <30 kDa. Although visualizing small peptides using SDS-PAGE is challenging, Tris-tricine gels afford better resolution. However, if you simply want to detect the peptide, MS remains the most accurate method for confirming the identity of a peptide.
Small peptides bind to Coomassie brilliant blue less readily than do larger proteins. Therefore, smaller peptides are difficult to detect using Coomassie or silver staining. The additional sample could be loaded to allow peptides to be visualized on gels; changing the percentage of the gel will only help if you think that your peptide migrated out the gel. In this instance, the percentage of crosslinker in a regular 17% gel could be increased, and the pH of the resolving gel could be increased to 9.5 (compared with the normal 8.8). Finally, the addition of 4–8 M urea helps sharpen bands.
The use of Western blotting rather than gel staining is a far more sensitive detection method. However, the peptide might simply pass through the membrane during transfer. If you think this occurs, the experiment can be repeated using two pieces of membrane and a shorter transfer time (<1 hour at 200 mA). A membrane with a 0.2-μm pore size should be sufficient: although smaller pore sizes are available, they should not be necessary. An additional option would be to try semi-dry transfer for 15–20 minutes using the current density (mA/cm2) recommended for the apparatus. A short transfer time of 15 min works for most small peptides. If it is possible to plan, a control small peptide labeled with biotin could be synthesized to monitor the transfer process and assess the ability of the peptide to bind to the membrane using streptavidin-conjugated HRP.
