Swartz:Protocols/Mass spec

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https://docs.google.com/document/d/1dM7bkfLRV6W1vNYTUsNuWry2B430t9VJWAHxQMWsKqw/edit

Proteins in polyacrylamide gel

  • Samples for protein identification by proteolytic digestion and LC-MS/MS are commonly submitted as Coomassie stained bands or spots in polyacrylamide gels.
  • The gels should be handled as little as possible, to minimize contamination by dust and keratin.
  • Destain Comassie stained gels by soaking for at least 2 hours in 10% acetic acid, 50% methanol, and 40% H2O with at least two changes of this solvent. If the gel still has a Coomassie *Blue background then continue destaining until the background is nearly clear.
  • Sypro Ruby and other fluorescent stained gels should not require an additional destaining step.
  • After destaining, soak in pure water until pH is neutral.
  • The spot or band of interest should be excised cleanly, excluding all of the surrounding blank gel; the goal is to maximize the ratio of protein to gel.
  • Cut the excised gel into small pieces (1-2 mm square) and placed in a clean eppendorf vial.
  • Wet samples should be shipped on dry ice. Dry gel pieces may be shipped at room temperature.

http://mass-spec.stanford.edu/SamplePrep.html#SamplePrepProteins Mudd Room 175

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