New England Biolabs, P8101S, Trypsin-ultra™, Mass Spectrometry Grade

Trypsin-ultra, Mass Spectrometry Grade is a serine endopeptidase, which selectively cleaves peptide bonds C-terminal to lysine and arginine residues.  Trypsin-ultra cleaves at Lys-Pro and Arg-Pro bonds at a much slower rate than when Lys and Arg are N-terminal to other residues. Related Categories Proteases,, Proteome Analysis Applications Glycomics and glycoproteomics,, Biotherapeutics and Antibody Analysis,, Protein Digestion Specification Reaction Conditions 1X Trypsin-ultra, Reaction Buffer Incubate at 37°C 1X Trypsin-ultra, Reaction Buffer 50 mM Tris-HCl 20 mM CaCl2 (pH 8 @ 25°C) Usage Concentration 100 ng/μl Molecular Weight Theoretical: 23675 daltons FAQ Q: Is there a compatible buffer for digesting with Trypsin (P8101S) and Endoproteinase GluC (P8100S) together? A: We would suggest a 50:50 mix of each enzyme's buffer. GluC works at maximal activity with a stimulating glutamic acid dipeptide in the reaction. Calcium chloride reduces autolysis of tryspin. Both buffers have a pH 8, Tris-HCI backbone. We have done our GluC/Trypsin digests this way with incubation overnight at 37°C. Neither enzyme is inhibited by the buffer mixing but they will, of course digest each other to some extent. Q: I have a very low concentration of protein and would prefer not to denature as a separate step with buffer exchange before digestion. What denaturants can he use in the Trypsin reaction itself? A: Trypsin cannot tolerate SDS but will tolerate low levels (>0.01%) of most non-ionic detergents. Also heating your protein to 95 C for 1 minute (if your protein remains soluble when heated) or putting it into urea or guanidinium, no more than 0.25 M for either, or you'll denature the Trypsin itself, should work. Q: I would like to use Trypsin to digest some proteins which are present in a cell culture supernatant. The supernatant is composed of DMEM supplemented with 0.2% BSA and antibiotics. Will the enzyme work in the cellular supernatant? The final reaction volume will be 100-300 ul. A: Trypsin will work in our included buffer but a digest can be done in most any buffer base (as long as no serine protease inhibitors are present) by adjusting the pH to 8-8.5 and adding CaCl2 to 10 mM. The calcium prevents autolysis of the enzyme. Other salt concentrations are not usually a problem. We generally use the Trypsin at a ratio of 1:20 to 1:100 enzyme:protein so you need an approximate protein concentration which could be determined by SDS PAGE or a Bradford dye protein assay. If the BSA at 0.2 % is the majority of the protein then this would be a 2 mg/ml or 200 ug/100 ul and require about 2 to 10 ug of Trypsin. Q: I am using Trypsin and am wondering about specificity. Does it cut at additional sites when in high concentration? A: No. Q: I want to know if calcium can be left out of the buffer since it is causing my DNA-protein complex to precipitate. A: Trypsin will self-proteolyze unless Ca is present. Q: I want to use Trypsin on 20 ug of protein. How much enzyme do I need to use? A: We have typically recommended a ratio by weight of 20:1 substrate to Trypsin, therefore in this case add 10 ul of 100 ng/ul Trypsin stock solution. For proteomics and large scale applications, we regularly use a 50:1 ratio. Some experimenters use ratios as high as 100:1 so as to limit Trypsin fragments as contaminant in the analysis. Care must be taken when using low amounts of Trypsin that a complete digestion is still obtained. A 10:1 ratio of sample: Tryspin would be the highest amount of trypsin that should be used. Q: How does one do a Trypsin in-gel digest? A: Use the Trypsin digestion buffer to re-swell the gel slice after it is dehydrated using acetonitrile, then add an amount of Trypsin to the sample in a ratio range of 1:10-20 wt./wt. enzyme:substrate. For example, a protein band estimated to be 1 ug would be combined with 50 to 100 ng of Trypsin and incubated at 25-37°C overnight. We generally clean-up our in-gel reactions on C18 ZipTips before MS (MALDI-MS or Ion Trap MS/MS). Q: Is there a simple way to remove Trypsin after protein cleavage? A: Bezamidine Sepharose (pH 8) will remove intact Trypsin, although some of the desired peptides may be lost in the process. The best strategy is to keep the ratio of substrate to trypsin, high, e.g. 50-100:1.