I am new to the enzyme and protein field of research. Therefore I am struggling with the following issues-
How to calculate the trypsin enzyme amount required for performing the complete hydrolysis of legume protein as substrate. The percentage of enzyme required with respect to protein, the amount of protein, and the ratio of enzyme: substrate? I also to want to know the analytical method for peptide analysis.
Hi, Sonia,
It partially depends on the quality of the trypsin you are using. For Promega trypsin, 1:50 or 1:100 trypsin to protein ratio worked for me. You also can do a titration of the trypsin amount and check on silver stained SDS-polyacrylamide gels if you see complete protein digestion.
Mass spec (MALDI or LC-ESI) would be an ideal technique to follow the digestion.
Debarati Bandyopadhyay Could do please tell me how to make 0.25% of Trypsin from 1:100 Trypsin?
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