Protein ID Solutions Sample Prep Guide
In-Gel Tryptic Digestion with Agilent Kit
3 Incubate sample at room temperature for 15 minutes.
4 Do one of the following:
For gel spots, add 25 µL digestion buffer to the tube.
For gel bands, add 50 µL digestion buffer to the tube.
5 Incubate sample at 37 °C for 4 hours or at 30 °C overnight with shaking.
6 If there is condensation on the lid of the tube, spin down so all solution is in
the bottom of the tube.
7 Remove digestion liquid and place in a clean tube.
8 (Optional) To further extract peptides, add 10 µL 1% trifluoroacetic acid
(TFA) or 1% formic acid solution to gel pieces and incubate for 5 minutes.
Remove extraction solution and add to mixture that you removed in step
If you are planning to desalt, use TFA because it helps with peptide
binding to the resin during cleanup.
This step also serves to inactivate trypsin, stopping additional enzymatic
A second extraction generally results in only a minor increase in peptide
9 If you elected to skip
, then add 1 µL of 1% TFA or 1% formic acid to
the digestion liquid. If you performed
, skip this step and go to
You need to add acid to inactivate the trypsin, so if you did not add it in
, then you need to add it here.
10 To prevent LC clogging or column damage, ensure sample is free of
11 Sample is now ready for liquid chromatographic separation and
electrospray ionization mass spectrometry (LC-ESI-MS). For
matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)
or 2D LC/MS/MS, additional processing/cleanup is required.
In general, use Agilent Peptide Cleanup C18 Pipette Tips (
If ammonium bicarbonate is the only salt in the sample and you plan to
perform SCX, skip the C18 cleanup and go to
N O T E
If 10 µL activated trypsin is insufficient to cover and fully swell gel pieces, be sure to
increase volume accordingly.