IN-GEL DIGESTION PROTOCOL

ABBREVIATIONS:

REAGENTS:

Alkylating Reagent:

TRYPSIN:

DESTAIN:

1. Remove any liquid from microfuge tube and use sealed pipette tip to cut gel slice into small pieces.
2. Add enough destain reagent to cover gel pieces. Shake at room temperature for 10 min. Discard liquid. If gels were stained using Sypro, go to REDUCTION/ALKYLATION FOR 1D GEL BANDS.
3. Repeat step 2 until gel pieces are clear. Usually 2 to 3 washes are necessary. If gel pieces still have blue color, rehydrate by adding 50 mM NH₄HCO₃ and shake for 10 min at room temperature. Discard liquid and repeat step 2.
4. For 2D spots (that have already been reduced and alkylated) you can proceed directly to step 8 in the next section. However, it will not hurt to reduce and alkylate again and may help your digestion.
   Notes: Destaining can be accelerated by sonicating in a heated (37° C) water bath during steps 2 and 3. For persistent blue color, add 100 mM NH₄HCO₃ and place in oven (50° C) for 10 minutes. However, the presence of a small amount of stain may not interfere with the mass spectral analysis and excessive heating is not recommended.
   

   REDUCTION/ALKYLATION

   (FOR 1D GEL BANDS):
   Purpose: In order to completely denature the protein we must reduce (DTT) disulfide bonds and alkylate (IAA) to prevent re-formation of bonds.
   1. 1. If not already dehydrated, dry gel pieces in Speedvac. Transfer the dried slices to clean tubes.
   2. 2. Add enough of the Reducing Reagent to fully cover gel pieces, taking into account the pieces will swell. Usually 50 µl is sufficient.
   3. 3. Incubate at 37° C for 1 hr.
   4. 4. During this time, prepare the Alkylating Reagent.
   5. 5. After incubating, cool to room temperature and remove excess Reducing Reagent. Add enough Alkylating Reagent to cover gel pieces. Place tubes in the dark and shake gently for 45 min.
   6. 6. Discard supernatant. Wash gel pieces with 100 mM NH4HCO3 for 10 min with shaking at room temperature. Discard supernatant.
   7. 7. Wash gel slices twice more using Destaining Reagent (10 min with shaking at room temperature and each time discarding supernatant).
   8. 8. Dehydrate gel pieces by placing in Speedvac. After drying, transfer the dried slices to clean tubes.
   9. 9. Add enough of a 0.2 µg/µl trypsin solution to swell the gels and incubate on ice or at 4°C for 15 min. Usually 20 µl is sufficient. The trypsin solution can be prepared and aliquoted ahead of time (20 microfuge tubes of 10 µl ea.) and stored in the freezer until ready. Discard unused trypsin.
   10. 10. Remove excess trypsin solution. Add 50 mM NH4HCO3/10% ACN, enough to cover, but not excess. Place in 37° C incubator.
   11. 11. Check pieces in 20 min, adding enough 50 mM NH4HCO3/10% ACN to keep pieces just covered.
   12. 12. Digest overnight (~19 hrs) at 37° C.
   
   
   

   EXTRACTION OF TRYPTIC FRAGMENTS

   Purpose: Extract peptides from gel prior to mass spec analysis
   
   1. 1. Remove microfuge tubes from incubator. Remove supernatant and save in clean microfuge tube.
   2. 2. Add enough 1.0% Formic acid/10% ACN to cover gel pieces. Shake for 10 min at room temperature (or sonicate), remove supernatant and combine with supernatant from previous step.
   3. 3. Add enough 50% ACN/01.0% Formic acid to cover gel slices and shake (or sonicate) for 10 min at room temperature.
   4. 4. Spin down and harvest supernatant with previous collections.
   5. 5. Place supernatant extract in Speedvac until concentrated to 10 µl. If extract goes to dryness (not good), add either 5 µl of 1.0% Formic acid (for LC/MS) or 5 µl of 1% Formic acid/10% ACN (for MALDI-MS) and vortex vigorously at room temperature.
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