Recommendation proteomics sample preparation in form of a PAGE gel band.

  • Use precast gels, or gels you have cast at least 24 hours before use.
  • Use <12% gels. If you need high resolution for low molecular weight proteins, use bis-tris gels and MES buffer. Higher % gels tend to lead to higher losses of proteins.
  • Do not use urea in the sample buffer.
  • Do not run samples with high-salt samples. Precipitate the proteins first, then reconstitute in sample buffer for tight bands.
  • If you use urea in the gel or running buffer, make it fresh just before use.
  • If the aim of the gel is to clean up incompatible solvents, detergents and salts, you can run it as a gel plug – allow the front just to enter the resolving gel and stop. All of the proteome will be in one band. Cut it and send it to us.
  • -> However, keep in mind there are other options to run your sample without the need to run on a gel.
  • If the aim of the gel is to identify specific bands, allow full resolution of the sample.
  • If you are excising bands based on Western blot, run short transfers and excise bands from the same gel!
  • If you expect the gel to stay intact for at least 2 hours before cutting, fix the gel – it will prevent sample diffusion. We strongly suggest to excise the bands ASAP.
  • Please use MS compatible stains – either silver stains or coomassie. Stains containing formaldehyde are incompatible with MS.
  • Do not freeze the gel band.
  • Bring each band in a separate tube, dry, without any added buffer.
  • As a general rule of thumb, the minimal protein amount for analysis is the one showing faint blue bands using GelCode Blue stain (~20ng).
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