Pooled saliva samples of different animals after initial profiling by SDS Gel undergone in solution trypsin digestion procedure. The expected digested samples again undergone SDS gel to check the status of digestion.The SDS gel shows undigested bands...Please Kindly elaborate and provide me the protocol or procedure to get peptides digested
Dear Laishram Kipjen Singh ,
Every protease has its own specific cleavage sites. Trypsin cleaves peptide chains mainly at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline. In vivo these peptide products are then further hydrolyzed into amino acids via other proteases.
Trypsin is because of its high proteolytic activity and cleavage specificity the most popular protease used in for example mass spectrometry (and subsequent protein/peptide identification).
Digestion with trypsin is rarely complete because for example tightly folded proteins resist proteolysis or because certain reagents used in protein preparation inhibit trypsin activity. You can add another enzyme to overcome this. See for example:
https://www.nature.com/articles/nmeth.f.371.pdf?origin=ppub
Or use another trypsin from another origin:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4076643/
If you really want to digest your sample up to individual amino acids than a combination with at least one more protease is necessary (for example chymotrypsin, which is orthogonal in its cleavage specificity and so on). See for more details and suggestions:
https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/febs.13287
PS. There are commercially available mixes of proteases often used in Food Science that might do want you want.
Hope this helps you somewhat further.
@ Dear Rob Kell thanks for your valuable and useful suggestion
You might also try heating the samples in boiling water after the digestion to help denature tightly-folded proteins, then add additional trypsin to continue the digestion.
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