UCSF In-Gel Digestion Protocol

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

1. 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.
2. 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)
3. Dice each gel slice into small pieces (1 mm2) and place into 0.65 mL siliconized tubes (PGC Scientific).
4. Add ~100μL (or enough to cover) of 25mM NH₄HCO₃/50% ACN and vortex for 10 min.
5. Using gel loading pipet tip, extract the supernatant and discard.
6. Repeat steps 3 and 4 once or twice.
7. 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.
   1. Prepare fresh solutions:
      10 mM DTT in 25 mM NH₄HCO₃ (1.5 mg/mL)
      55 mM iodoacetamide in 25 mM NH₄HCO₃ (10 mg/mL)
   2. 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.
   3. 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.
   4. Remove supernatant (discard). Wash gels with ~100 μl NH₄HCO₃, vortex 10 min, spin.
   5. 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.
   6. Speed Vac the gel pieces to complete dryness (~20 min). Proceed with trypsin digest.
8. 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.
9. Rehydrate the gel pieces on ice or at 4°C for 10 min. Spin. Add 25mM NH₄HCO₃ as needed to cover the gel pieces.
10. Spin briefly and incubate at 37°C for 4 hours - overnight.

Extraction of Peptides

1. Transfer the digest solution (aqueous extraction) into a clean 0.65 mL siliconized tube.
2. To the gel pieces, add 30 μL (enough to cover) of 50% ACN/5% formic acid, vortex 20-30min., spin, sonicate 5 min. Repeat.
3. Vortex the extracted digests, spin and Speed Vac to reduce volume to 10 μL.
4. 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.
5. 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.

References:
Rosenfeld, et al., Anal. Biochem. (1992) 203(1), 173-179. [Pubmed]
Hellman, et al., Anal. Biochem. (1995) 224(1), 451-455.[Pubmed]