The Keratin Issue
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Ethanol Precipitation with Glycogen as Carrier
If there is visible precipitate in total mixture before centrifugation, it means: a. there was too much salt in the protein solution; b. some amount of the protein(-s) will be lost due to insolubility.
To avoid this problem:
TCA Precipitation with Deoxycholate as Carrier
For in-solution digests and subsequent mass spectrometric analysis, the sample should be free of ANY detergents (SDS, Triton X100, NP-40, etc.pp.). Once that is insured, the following protocol can be used:
If denaturing, reduction of the SS-bonds, and alkylation of the free thiol groups is necessary, we suggest the following protocol:
Alternatively, following protocol can be used:
There are numerous staining methods available that are compatible with subsequent proteolytic digestion and mass spectrometric analysis. Most of them are based on Coomassie staining, silver staining or using fluorescence dyes such as Sypro Ruby or Sypro Orange. Whereas Coomassie staining is simple and cheap, the sensitivity is sometimes insufficient. Silver staining overcomes the sensitivity limitations of Coomasssie staining, but is rather cumbersome. The ease of Coomassie staining and the sensitivity of silver staining are combined in fluorescence Sypro dyes. Those, however, have the disadvantage that they are rather expensive, a fluorescence scanner is a prerequisite for imaging, and excising the gel plugs/bands of interest requires portable UV sources. Irrespective of the staining method used, it is crucial to avoid irreversibly fixing the proteins in the gel during staining; this consideration especially applies to some 'traditional' silver staining protocols that use glutaraldehyde or formaldehyde for fixing and developing which will render the gels incompatible with subsequent mass spectrometric analysis. A variety of mass spec compatible silver staining protocols are given below.
We have good experience with numerous commercial Coomassie staining kits including GelCode Blue Stain from Pierce, Bio-Rad's Bio-Safe Coomassie stain and the Colloidal Blue Staining kit from Invitrogen. A general Coomassie staining protocol is given below. If commercial kits are used, we strongly suggest following the manufacturer's protocols.
SYPRO Ruby Staining
Filtration, Desalting and Pre-concentration Using Micro-scale Reversed Phase Columns (StaGE tips)