60
Protein ID Solutions Sample Prep Guide
6
In-Gel Tryptic Digestion with Agilent Kit
To minimize freeze-thaw cycles and to increase storage stability, divide the
hydrated trypsin into four separate tubes of
~5 µL each. Store each aliquot at
-20 °C in a non-frost-free freezer. This solution is used to prepare the trypsin
intermediate solution as needed (see below).
Trypsin intermediate solution
When required, thaw a trypsin stock aliquot on
ice. Dilute stock 10-fold by adding 45 µL ultrapure water. This solution may be
stored at -20 °C for 2 months without significant loss of activity.
Destaining solution
Mix 80 mg of ammonium bicarbonate with 20 mL of
acetonitrile (ACN) and 20 mL of ultrapure water. The destaining solution may
be stored at 4 °C for 2 months. This stock solution is sufficient for 50 to 100
digestions and can be prepared three times with this kit.
Digestion buffer
Mix 10 mg of ammonium bicarbonate with 5 mL of ultrapure
water (final concentration
~25 mM). Digestion buffer may be stored at 4 °C for
2 months. This stock solution can be prepared three times with this kit.
Prepare just before use:
Reducing buffer
Prepare just before use as described in
step 1
of the
"Reduction and alkylation (optional)"
procedure. Mix 3.3 µL of TCEP with
30 µL of digestion buffer for each digest to be performed. Final TCEP
concentration is
~50 mM.
N O T E
An excess of digestion buffer is supplied to minimize the need for long-term storage and
weighing minute quantities of ammonium bicarbonate.
Table 11
Preparation of reducing buffer
Number of samples
Amount of TCEP (µL)
Amount of digestion buffer (µL)
1
3.3
30
10
33
300
20
66
600
30
99
900