Quick (relatively) Method for Preparing Gel Fragments for Mass Spec
Materials:
Acetonitrile (100% HPLC Grade) (ACN)
1 M Ammonium BiCarbonate (10X ABC)
100 mM ABC (pH 8-8.5 if made fresh)
2:1 100 mM ABC : ACN
10 mM DTT solution (10 uL of 1M DTT + 990 uL 100 mM ABC)
55 mM Iodoacetamide
solution (100 uL of
550 mM Iodoacetamide + 900 uL of 100 mM ABC)
Trypsin (1 vial of Trypsin in 100 mM ABC of appropriate volume per sample)
1.) Cut up gel slice into pieces < 1 mm on a side and put in 2 mL tube
2.) Add 2:1 ABC : ACN and incubate 20 min at 25C and 750 rpm
3.) Discard super
4.) Repeat steps 2 & 3 (remove coomassie blue stain)
5.) Add ACN and incubate 5 min at 25C and 750 rpm
6.) Discard super
7.) Add 10 mM DTT Solution and incubate 1 hour at 30C and 750 rpm
8.) Discard super
9.) Add ACN and incubate 5 min at 25C and 750 rpm
10.) Discard super
11.) Add Iodoacetamide Solution and incubate in the dark (drawer) for 30 min
12.) Discard super
13.) Add ACN and incubate 5 min at 25C and 750 rpm
14.) Discard super
15.) Add 2:1 ABC : ACN and incubate 30 min at 25C and 750 rpm
16.) Discard super
17.) Repeat steps 14 and 15 (wash: remove last traces of coomassie blue stain)
18.) Add ACN and incubate 5 min at 25C and 750 rpm
19.) Discard super
20.) Add Trypsin and incubate 15 min on ice
21.) Put samples in 37C overnight
22.) Remove super from Overnight digestion and put in new tube
23.) Add 2:1 ABC : ACN and incubate for 20 min at 25C and 750 rpm
24.) Remove super and pool with digestion super
25.) Add ACN and incubate 5 min at 25C and 750 rpm
26.) Remove super and pool with digestion super
27.) Place samples in speed vac and run until dry
28.) Bring up in 100 uL of sample buffer each and transfer to Orbi-Trap or LTQ sample bottles.
29.) Setup run on machine and load samples.
30.) Check results and submit searches