Improve Research Reproducibility A Bio-protocol resource
bpdetail_icon_lock
Preprint

Trypsin digestion

MD Meng-Qiu Dong

Last updated date: Sep 1, 2021 Views: 718 Forks: 0

original An abbreviated version of this protocol was published in eLife in Mar, 2016
Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states
download pdf Download PDF
ask Ask a question
cite How to cite
nfavorite Favorite

                                                              

  1. dissolve proteins in 20 ul of 8 M urea, 100 mM Tris pH 8.5 (a minimum of 10 ul for minute amount of proteins). 
    Sonicate for 10 min.
  2. add 0.2 ul 0.5 M TCEP (5 mM final conc.). Incubate at RT for 20 min. (note: take a 5-ul aliquot of the 0.5 M TCEP stock stored at -80 ºC and toss after each use)
  3. add 0.4 ul 0.5 M iodoacetamide (10 mM final conc.) and incubate at RT for 15 min in the dark. (note: make a stock 0.046 g/500 ul H2O, and store 5-ul aliquots at -80 ºC. Toss after each use).
  4. Trypsin Digest
  1. Dilute sample by a factor of 4 with 100 mM Tris pH 8.5 (i.e. add 60 ul of Tris to 20 ul of sample, final vol = 80 ul, final conc. of urea = 2 M)
  2. add 100 mM CaCl2 (i.e. add 0.8 ul) to a final conc. of 1 mM to enhance the activity of trypsin and increase its stability.
  3. add methylamine to 20 mM to reduce carbamylation.  [1.2 M CH3NH2 solution (60x) can be found in microfuge tubes along with the 40% stock solution (~11.6 M or 580x) in the hood]
  4. add 1 ul of 0.5 ug/ul trypsin (or at a 100:1 substrate:enzyme ratio if you know the amount of proteins)
  5. overnight incubation at 37 ºC in the dark (note: avoid evaporation or condensation on the lid, which would increase the concentration of urea and everything else in the sample tube. This means that the lid needs to be sealed tight and kept warm.).
  6. Add 5 ul 90% formic acid (~5% final conc.), spin in a tabletop centrifuge at  13,000 rpm for 10-20 min, transfer the supernatant to a new tube, and freeze at –80 degree.
Copyright: Content may be subjected to copyright.
How to cite：
Readers should cite both the Bio-protocol preprint and the original research article where this protocol was used:
  1. Dong, M(2021). Trypsin digestion. Bio-protocol Preprint. bio-protocol.org/prep1359.
  2. Tan, D., Li, Q., Zhang, M., Liu, C., Ma, C., Zhang, P., Ding, Y., Fan, S., Tao, L., Yang, B., Li, X., Ma, S., Liu, J., Feng, B., Liu, X., Wang, H., He, S., Gao, N., Ye, K., Dong, M. and Lei, X.(2016). Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states. eLife. DOI: 10.7554/eLife.12509

Do you have any questions about this protocol?

Post your question to gather feedback from the community. We will also invite the authors of this article to respond.

post Post a Question
0 Q&A
This protocol preprint was submitted via the "Request a Protocol" track.