Proteomics Core FacilityProtocols

Gel staining prior to protein identification

We accept samples for protein identification from coomassie and silverstained gels. Coomassie staining is preferred, so only use silverstaining if the amount of protein is insufficient for visualization by coomassie.

Coomassie staining

Not all coomassie stains are compatible with mass spectrometry.  We strongly recommend that you mix your own coomassie stains (see protocol below for the recipe).  We DO NOT accept samples that have been stained with any commercial 'instant' or 'ready-to-use' staining kits.   We also DO NOT accept gels that have been boiled or microwaved to speed up the staining or destaining process.  Please NEVER use overhead projector foils for gel scanning as this prevents the gel from being used for MS experiments.

A protocol for colloidal coomassie staining can be downloaded here.

Silver staining

Only some silver staining protocols are compatible with mass spectrometry. DO NOT use commercial kits, we only accept samples that have been stained using this protocol.

Cutting of gel bands

If you have gels from which you like individual bands to be identified by MS, you have two options. First, you can send (or bring) the gel to the facility, and we cut out the band(s) that you indicate.

Alternatively, you can cut out gel bands yourself. In this second scenario, please take precautions to prevent contamination (keratins, etc).

Please use the following guidelines.  

 

Mass determination of intact proteins

The molecular mass of proteins is best determined under denaturing conditions. Please observe the following guidelines for sample preparation:

  • Provide at least 10 µL of a protein at a concentration of >1 mg/ml
  • Avoid the use of detergents
  • Samples should contain less than 2% glycerol
  • Proteins should be as pure as possible - we can measure simple mixtures of a few proteins or where both a modified and non-modified version of the protein exist, but not complex mixtures.  A gel picture can be sent to Facility staff ahead of a request to evaluate whether the sample is pure enough for a succesful analysis.

Only in very specific cases we can analyse proteins under non-denaturing (or native) conditions.

  • Provide at least 100 µl of a protein at a concentration of >2 mg/ml
  • The protein must be in a volatile buffer, preferably 25 mM ammoniumacetate at any pH below 7. Plain 25 mM ammoniumacetate pH = 6.7, for pH adjustment use acetic acid.
  • Avoid the use of detergents
  • Sample must be salt-free