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- Gel Running
We accept gel spots/bands from all types of SDS-PAGE gels and 2D gels. Users should pay special attention to reduce contamination, such as keratin. Always wear gloves when handling gels and wash apparatus thoroughly. It is advised to use fresh sample loading buffer, running buffer, fixing and staining solutions with clean DI water.
- Gel Staining
Coomassie blue based staining (traditional, Colloidal blue, SimplyBlue, etc.) and fluorescent dye staining (such as Molecular Probes' SYPRO Ruby staining) are all compatible to mass spec based protein ID.
Silver staining is also acceptable as long as you use mass spectrometry compatible protocols. We generally find good results using PIERCE Silver SNAP or Invitrogen silver stain kit. Please note that we found de-staining is not necessary and may have negative effect on mass spectrometry results. We also noticed that the developing step of the staining procedure is critical. Please stop the developing reaction the moment your band is visible. It is not advised to develop more than 10 min.
- Band Cutting
Take a photograph of the gel before cut it! Once you cut out a band, please mark the position on the photograph. Please submit the picture with your sample. The gel picture will help us in designing the sample loading amount and sequence to avoid carry-over.
Put the gel on a clean glass plate and cut the bands with sharp razor blades (use new razor blade for each gel band to avoid cross-contamination). Do your best to avoid any direct contact with the gel with bare hands, light box, or any other possible sources of keratin contamination.
Try to cut out a band as close to your protein as possible. Higher protein/gel ratio ensures more efficient peptide extraction, higher MS detection sensitivity, therefore better protein ID results.
Put each gel band in a clean, 0.5 or 1.5 ml micro-centrifuge tube, and label each tube carefully. Follow our sample submission procedure to submit your samples.
Duplicate samples are always welcome although generally not necessary. They will be used as backup and will not be double-charged.
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