Proteins separated by gel electrophoresis

Use pre-cast gels

If your proteins are to be separated by gel electrophoresis before MS-analysis, please use pre-cast Invitrogen/NuPAGE Bis-Tris SDS-PAGE gels instead of casting them yourself.

Use MS-compatible staining method

Not all gel staining methods are compatible with MS-analysis. While all types of Coomassie staining are compatible, silver staining applying glutaraldehyde in the sensitizing solution is not compatible with MS. Therefore, it is necessary that the recommended silver staining procedure (see Protocols, silver stain protocol according to Blum) or a MS-compatible silver staining kit is used (e.g. Pierce Silver Stain Kit 24600, Invitrogen SilverQuestKit LC6070). For silver staining, do not overstain the gel, as this reduces the yield of identifiable peptides from your sample. For Coomassie staining, also try to only stain your gel for the minimum time to (just) detect your protein(s) of interest. Extended staining will increase the background in subsequent MS analysis. Destain the gel thoroughly to clear the background and to enhance visibility of the band. Do NOT heat your gel in the microwave to speed up the staining!

Avoid keratin contaminations

It is essential to prevent contaminations of the gel by hair and skin particles or cloth threads, therefore carry out all staining steps in closed dishes and use filter-sterilized solutions which are dedicated to MS-experiments. Wear gloves and lab coats at each step. Be aware that any surfaces, glassware or chemicals exposed to the lab atmosphere for more than a few minutes will be contaminated with keratin as dust from the atmosphere settles. Therefore wash everything (gel tank, staining trays...) before use and then keep it covered.

Provide a picture of the gel and all known information about the sample

Please provide a scan of the stained gel, where the bands of interest are labeled. Additionally, it is important that you provide all known facts about the nature of your sample, helping us to choose the optimal digestion and analysis strategy. The protein bands to be analyzed by MS will be cut out in a laminar flow hood in the MS-lab by a member of the MS-unit. In case you want to have many bands analyzed (i.e. more than 15), you may be asked to cut them out yourself and to help with the digest in our laboratory. This also depends on the total number of samples that are waiting to be processed at that moment.