Protein ID Solutions Sample Prep Guide
In-Gel Tryptic Digestion with Agilent Kit
Trypsin is a serine protease that specifically cleaves peptide bonds at the
carboxyl side of lysine and arginine residues; however, cleavage can be blocked
or slowed by proximal acidic, aromatic, or proline residues with proline having
the most significant effect. Peptide fragments with one missed cleavage are
common and should be taken into consideration during mass analysis.
The modified trypsin provided in this kit displays only limited autolytic
activity that should not interfere with mass spectral analysis. Trypsin
fragments of mass 842.51 (m/z, [M+H]
) and 2211.10 (m/z, [M+H]
) will be the
most common using standard conditions and can be used as internal
The Protein In-Gel Tryptic Digestion Kit is designed for colloidal Coomassie or
fluorescent dye-stained acrylamide gel slices. For protein bands stained with
MS-compatible silver stains or reversible zinc staining, alternative destaining
procedures will be required [3, 4].
Increase incubation time.
Ensure gel slice was dry before addition of enzyme. A dry gel slice will
pull trypsin into gel slice and increase hydration volume.
Enzyme is losing activity
Use a new trypsin stock aliquot.
Ensure gel slice has been completely destained and trypsin working
solution has been diluted with digestion buffer.
Ensure gel slice has been completely destained.
Poor mass spectrum
detection limits of
Ensure sample is within the detection limit of the specific downstream
application; concentrate digest with Cleanup C18 Pipette Tips
Note: Limits vary considerably based on application and
Clean up digest with Cleanup C18 Pipette Tips (5188-5239).