1. Protein amount. Our general rule of thumb is that if there is enough protein to be strongly stained with Coomassie G-250, there should be enough for Mass Spectrometry analysis. We prefer that you scale up your prep so that you have 10-20 ng of protein in the gel band. Please do not scale up by sending multiple gel pieces, as this increases gel volume and decreases the efficiency of peptide extraction from the gel. Instead, load a higher amount of protein into ONE lane of the gel.

2. Minimize gel volume. It is important to minimize the gel volume and maximize protein concentration. As such, we recommend you cut out only the area with your protein of interest (1x1x2mm size) and exclude as much unstained gel as possible.

3. Silver stained gel band should be compatible with Mass Spectrometry. You need to make sure your silver staining solution does not contain aldehyde that cross-links your proteins to the gel matrix, resulting in low extraction efficiency. Non-formaldehyde Silver Stain uses carbohydrazide instead of formaldehyde for reduction/development.

4. Reduction/alkylation. The gel pieces can be reduced and alkylated to improve tryptic digestion and produce more peptides for Mass Spectrometry analysis. This procedure is especially important for gel bands with low protein concentrations, that is, bands that stain faintly with Coomassie or bands that can only be visualized by silver staining. Please treat your samples by using the recommended protocol (Shevchendo et al, Natural Protocol, 2006, vol1: 2856) before shipping to us.

5. Shipping condition. When you are ready to send your sample, please add HPLC-grade water to cover the excised gel band in a 1.5ml eppendorf tube, then send on ice pack. Please avoid sending samples on Friday, as they will have to sit in a warehouse over the weekend.

Please click here to view the experimental procedure for Protein ID by Mass Spectrometry.


Call our tech support at 510-887-0889 or send email to support@appliedbiomics.com with any questions.