This is the current procedure for In-Gel digests used at
UCSF (as of 2002.04.08). It has been refined to its current state by contributions
from lab members over the last few years.
In-Gel Digest Procedure
- 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 blades with methanol-soaked lint-free cloth.
- Prepare the following solutions:
25 mM NH4HCO3 (100 mg/50 ml)
25 mM NH4HCO3 in 50% ACN
50% ACN/5% formic acid (may substitute TFA or acetic acid)
12.5 ng/μL trypsin in 25mM NH4HCO3 (freshly diluted)
- Dice each gel slice into small pieces (1 mm2) and place into 0.65
mL siliconized tubes (PGC Scientific).
- Add ~100μL (or enough to cover) of 25mM NH4HCO3/50% ACN and vortex
for 10 min.
- Using gel loading pipet tip, extract the supernatant and discard.
- Repeat steps 3 and 4 once or twice.
- Speed Vac the gel pieces to complete dryness (~ 20 min).
For low-level proteins (<1 pmol), especially those separated
by 1-D SDS-PAGE, reduction and alkylation is recommended. These procedures
are performed after step 6.
- Prepare fresh solutions:
10 mM DTT in 25 mM NH4HCO3 (1.5 mg/mL)
55 mM iodoacetamide in 25 mM NH4HCO3 (10 mg/mL)
- Add 25 μL (or enough to cover) 10 mM DTT in 25 mM NH4HCO3 to
dried gels. Vortex and spin briefly. Allow reaction to proceed at
56°C for 1 hr.
- Remove supernatant, add 25 μl 55 mM iodoacetamide to the gel
pieces. Vortex and spin briefly. Allow reaction to proceed in the
dark for 45 min. at room temperature.
- Remove supernatant (discard). Wash gels with ~100 μl NH4HCO3,
vortex 10 min, spin.
- Remove supernatant (discard). Dehydrate gels with ~100μL (or
enough to cover) of 25 mM NH4HCO3 in 50% ACN, vortex 5 min, spin.
Repeat one time.
- Speed Vac the gel pieces to complete dryness (~20 min). Proceed
with trypsin digest.
- Add trypsin solution to just barely cover the gel pieces. Estimate
the gel volume and add about 3x volume of trypsin solution. This volume
will vary from sample to sample, but on average ~5-25 μL is sufficient.
- Rehydrate the gel pieces on ice or at 4°C for 10 min. Spin. Add
25mM NH4HCO3 as needed to cover the gel pieces.
- Spin briefly and incubate at 37°C for 4 hours - overnight.
Extraction of Peptides
- Transfer the digest solution (aqueous extraction) into a clean 0.65
mL siliconized tube.
- To the gel pieces, add 30 μL (enough to cover) of 50% ACN/5% formic
acid, vortex 20-30min., spin, sonicate 5 min. Repeat.
- Vortex the extracted digests, spin and Speed Vac to reduce volume
to 10 μL.
- Either proceed with C18 ZipTip (Millipore) cleanup or analyze with
LC-MS. Add 2-5 μL of 5% formic acid. When analyzing low levels
of protein, concentrate the petides by eluting from ZipTips using 3μL
of elution solution, into a clean 0.65 mL siliconized tube.
- Use 1μL of the unseparated digests for analysis by MALDI.
Matrices for unseparated digests:
a-cyano-4-hydroxycinammic acid in 50% ACN/1% TFA (10 mg/mL).
2,5-dihydroxybenzoic acid (DHB), saturated solution in water.
Rosenfeld, et al., Anal. Biochem. (1992) 203(1), 173-179. [Pubmed]
Hellman, et al., Anal. Biochem. (1995) 224(1), 451-455.[Pubmed]