In-Gel Digestion Protocol
Wearing gloves and sleeve protectors, wipe down ALL surfaces in the hood with
methanol/water moistened lint-free cloth, including the outside of all your tubes
(make sure to not wipe off the labeling!), the outside and inside of the Speed Vac and
centrifuge, tube racks, bottles etc. Wipe razor blade (scalpel) with methanol-soaked
lint-free cloth. Use highest grade purified solvents.
Prepare the following solutions:
5% acetic acid in 50% AcN – overnight washing solution (WS0)
25 mM NH4HCO3 (40 mg/20 mL) – washing solution #1 (WS1) – store at 40C for up
to 8 weeks
25 mM NH4HCO3 /AcN (1:1) – washing solution #2 (WS2) – store at 40C for up to 8
weeks
100% AcN – washing solution #3 (WS3)
5% formic acid (FA) in 50% AcN – extraction buffer (EB)
10 mM DTT in 25 mM NH4HCO3 (1.5 mg/mL) – fresh only!
55 mM (20 mg/mL) iodoacetamide (IAA) in 25 mM NH4 HCO3– fresh only!
25 ng/µL Trypsin Gold (Sigma) solution in 50 mM acetic acid – store only at -800C
as 5 µL aliquots (one tube can be re-frozen 3 times). Note: Once trypsin is activated
by risen pH in 25mM NH4HCO3 it should be used or disposed.
Washing:
1. Dice each gel slice into small pieces (~1 mm2) and place into small glass vial
2. Add 200μL WS0, vortex, incubate 10 min
3. Using gel loading tip, extract the supernatant and discard
4. Add 200μL WS0, vortex, incubate overnight
5. Using gel loading tip, extract the supernatant and discard
6. Add 200μL WS1, vortex, incubate 10 min
7. Using gel loading tip, extract the supernatant and discard
8. Repeat the washing with WS1 twice.
9. Add 200μL WS2, vortex, incubate 10 min
10. Using gel loading tip, extract the supernatant and discard
11. Repeat the washing with WS2 twice.
12. Add 200μL WS3 and vortex, incubate 5 min
13. Speed Vac the gel pieces to complete dryness (10 min).
Reduction/Alkylation:
14. Add 100 μL (enough to cover) 10 mM DTT, vortex, incubate 60 min
15. Discard supernatant; add 100 μL 55 mM IAA vortex, incubate 60 min in dark
16. Discard supernatant, wash gels with 200 μL WS1, vortex, incubate 10 min
17. Discard supernatant, wash gels with 200μL WS2, vortex, incubate 10 min
18. Repeat the wash with WS2 once.
19. Add 100μL (enough to cover) WS3 and vortex, incubate 5 min
20. Speed Vac the gel pieces to complete dryness (10 min).
Digestion:
21. Add freshly diluted trypsin solution (do not re-freeze trypsin aliquots) to just
barely cover the gel pieces. Add at least 3-times more than volume of the gel.
This volume should be 25 ÷ 50 µL. Dilute frozen trypsin solution at least 1:10
(v./v.) in 25mM NH4HCO3 (WS1). Final trypsin concentration should not be
higher than 10 ng/µL. Typically, one should have 1:10 (trypsin-protein) mixture.*
22. Rehydrate the gel pieces on ice or at 4°C for 10 min.
23. Discard trypsin solution, and add 1-2 drops of WS1 to cover the gel pieces. That
will prevent gel drying during overnight incubation.
24. Incubate at 37°C overnight (or at least 4 hours).
Extraction of Peptides:
25. Transfer the digest solution (if any left) into a clean glass vial
26. To the gel pieces, add 20 μL (or enough to cover) of EB, vortex, wait 10 min
27. Repeat extraction twice.
28. Vortex the extracted digests, spin and Speed Vac to dry completely (10 min)
29. Bring to the Proteomics Facility immediately.
References:
Rosenfeld, et al., Anal. Biochem. (1992) 203(1), 173-179. [Pubmed]
Hellman, et al., Anal. Biochem. (1995) 224(1), 451-455.[Pubmed]
* - First, estimate the amount of protein in the Coomassie-stained gel spot visually.
Depending on the MW it might contain up to 100 pmoles (typically 1 ÷ 10 pmoles).
Porcine pancreatic trypsin MW ~ 24 kDa; 1 pmole is about 24 ng. For instance, if you
estimated your protein spot as 10 pmoles, you need 2 pmoles (~ 50 ng) of trypsin.
Thus, mix 2 µL of 25 ng/µL trypsin solution to 23 µL 25mM NH4HCO3 and add to
the gel.
Zip-Tip Procedure
We recommend purifying and concentrating the final peptide mixture by using ZipTip C18 micro-tips. Zip-Tips are disposable; used with a standard 10 μL pipette; one
tip is enough to purify few tens of picomoles of a peptide mixture.
Prepare the following solutions:
0.1% acetic (or formic) acid in MS grade purity water (buffer A)
0.1% acetic (or formic) acid in MS grade purity AcN (buffer B)
5% formic acid (FA) in 50% AcN – extraction buffer (EB)
Purification Protocol:
1. Conditioning: buffer B; 2 times (up & down, dispense)
2. Equilibrating: buffer A; 2 times (up & down, dispense)
3. Sample: your digest in 25mM NH4HCO3 (WS1) right after trypsinization; if you
have a dried sample – dissolve in 20 μL buffer A
4. Bind peptides: pipet your sample up & down; 5-10 cycles
5. Wash peptides: buffer A; 3 times (up & down, dispense)
6. Elute peptides: place the Zip-Tip inside glass micro-tube or insert, drop 10 μL of
EB into the Zip-Tip, attach pipette and elute EB through the Zip-Tip, repeat this
step using buffer B;
7. Speed Vac the eluted mixture to dry completely (10 min)
8. Bring to the Proteomics Facility immediately.