Aug 05, 2020
Trypsin digestion
- 1domnic colvin
- UM-DAE Centre for Excellence in Basic Sciences
Document Citation: avinash.kale 2020. Trypsin digestion. protocols.io https://dx.doi.org/10.17504/protocols.io.bjdpki5n
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: August 05, 2020
Last Modified: August 05, 2020
Document Integer ID: 40079
Trypsin digestion
In solution trypsin digestion of the TRIzol extracted protein sample was carried out by using Trypsin (Promega). Briefly, 15 µL of Digestion Buffer (50 mM Ammonium bicarbonate) and 1.5µL of Reducing Buffer (100mM Dithiothreitol) were added to 10uL of the protein solution to the micro-centrifuge tube and the final volume was adjusted to 27 µL with ultrapure water. The samples were incubated at 56 °C for 45 minutes and allowed to cool at room temperature. 3 µL of freshly prepared alkylation buffer (100mM Iodoacetamide) was then added to the tube and kept for incubation in the dark at room temperature for 20 minutes. Finally, 10 µL of Trypsin (20µg/ml) solution was added in the tube and kept for incubation at 37°C for 15 hours (overnight)(25).