Does your sample contain proteins of interest that are <20 kDa? Please download a protocol on how to detect synthetic peptides using SDS-PAGE.
Tricine-SDS-PAGE is commonly used to separate proteins in the mass range of 1-100 kDa. It is the preferred electrophoretic system for the resolution of proteins smaller than 30 kDa.
It is indeed very difficult to see the small peptide by SDS-PAGE. Tris-tricine gel will give you a better resolution. If you just want to detect the peptide, Mass Spec is still the best way to confirm the peptide identity.
Small peptide binds less Coomassie brilliant blue than larger protein. Thus smaller peptides are harder to detect by coomassie staining or silver staining. If you really want to see your peptide on the gel, you can try to load more samples. Changing the gel percentage won’t help much unless you think your peptide migrated out of the gel. You can increase the percentage of cross linker in the regular 17% gel. In addition increase the pH of your resolving gel to 9.5 as compared to your regular 8.8. Plus, the addition of urea (4-8M) helps sharpen bands.
If you are going to use western, which is a way more sensitive detection method, please use Western instead of the gel staining. However the peptide may simply pass through the membrane. If you repeat the experiment, try to put two pieces of membrane and shorter time of transfer (less than 1 hour at 200 mA). 0.2um pore could be enough. You can get smaller pore but that shouldn’t be necessary. You may want to try semi-dry transfer for 15-20 minutes at the recommended current density (mA/cm2) for the apparatus. A short 15 min transfer time works for most of the small peptides.
If you can plan ahead and synthesize a control small peptide labeled with biotin, you can monitor the transfer process and its ability to bind the membrane with streptavidin-conjugated HRP.
Please download this protocol for Tricine-SDS-PAGE, which includes efficient methods for Coomassie blue staining, silver staining, and electroblotting.
Download the Protocol
- To download the protocol, click the download button below.