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Dear all, does anyone has experience of how long I have to incubate a protein sample with trypsin for a complete digestion? I incubated o.n. at 37°C. Is this generally enough time (independent on trypsin concentration)? And does anyone know if trypsin needs on the left and on the right of the cleavage site some amino acids to work well? Especially for the case of two or more successive cleavage sites? Thank you all very much. Paul |
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I think there is no answer to this, you will have to use trial-and-error. Some proteins are just more protease-resistant than others. You will have to perform small-scale trials with different times and trypsin to protein ratios and then scale up. You may find your site is resistant and uncleavable, alternatively you may find an unexpected internal site that gets cleaved more efficiently than the desired site. For the second scenario there is no solution I can think of apart from site-directed mutagenesis to remove the undesired site. In the first, you may try partial unfolding of the protein at higher than 37 ºC temperatures, followed by trypsinisation at 37 ºC. |
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