Mass Spectrometry Facility - In-Gel Digest Procedure
This is the current procedure for In-Gel digests used at UCSF (as of 04/08/02). 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/ÂµL 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 ~100ÂµL (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 NH4 HCO3 (10 mg/mL)
- Add 25 ÂµL (or enough to cover) 10 mM DTT in 25 mM NH4HCO3 to dried gels. Vortex and spin briefly. Allow reaction to proceed at 56 Â°C for 1 hr.
- Remove supernatant, add 25 Âµl 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 Âµl NH4 HCO3, vortex 10 min, spin.
- Remove supernatant (discard). Dehydrate gels with ~100ÂµL (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.
- Prepare fresh solutions:
- 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 ÂµL is sufficient.
- Rehydrate the gel pieces on ice or at 4Â°C for 10 min. Spin. Add 25mM NH4HCO3 as needed to cover the gel pieces.
- Spin briefly and incubate at 37Â°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 ÂµL (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 ÂµL.
- Either proceed with C18 ZipTip (Millipore) cleanup or analyze with LC-MS. Add 2-5 ÂµL of 5% formic acid.
- Utilize 1ÂµL 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, 173-179.
- Hellman, et al., Anal. Biochem. (1995) 224, 451-455.
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