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43 Gortner Laboratory
1479 Gortner Ave.
St. Paul, MN 55108
Phone: (612) 625-2280
Fax: (612) 625-5780

46 Gortner Laboratory
Phone: (612) 625-6283

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Minneapolis, MN 55455
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or (612) 624-2674
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In-Gel Digestion Protocol

• Wear gloves to minimize contamination from keratins
• Siliconized tubes are highly recommended for proteolytic digestions
• Use small bore pipette tips such as gel loading tips
• Note: Excise a blank spot that does not contain protein, and process in the same manner as excised bands. A MW marker or other protein of your choice can be used for a positive control.

Silver Destaining

1. Excise the spot/band and place in very clean water (such as Mill-Q water) in a siliconized microcentrifuge tube.  Discard water.
   
   
2. De-stain (remove silver stain) spot with a 1:1 solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulphate made FRESHLY before use. Use 300 – 500 uL, incubate for 8 minutes, and discard.
   
   
3. Add 100 uL of the destaining solution if necessary, incubate for 1 minute and discard.
   
   
4. Wash 4 x with 1 mL of Mill-Q water, 8 minutes per wash.
   
   
5. Add 100% acetonitrile for 15 minutes, and discard solution
   
   
6. Speed-Vac for approximately 30 minutes (or less, until dry).

   [Coomassie Destaining: See separate protocol for destaining and then continue here:]
   [If Cysteine Reduction/Alkylation is desired, see Protocol at end of page then continue here.]
   

7. Add Sequence-Grade Modified Porcine Trypsin (Promega, for instance) to get a final concentration of 12.5 ng/uL (ng/microliter) trypsin, and a final concentration of ice-cold (4 °C) 50 mM ammonium bicarbonate (relatively fresh), 5 mM CaCl₂, pH between 8—9, and add enough solution to the spots so they are just covered.  Incubate on ice for 1 hour.  Check the spots every 10 minutes to insure that they have not absorbed all of the liquid. If the liquid is absorbed, add more of the trypsin/biocarb solution (see Note, below).

   NOTE: Do not use >1 ug of trypsin (total protein amount) per sample, that is, not >80 ul of a 12.5 ng/ul solution

8. After 1 hour, discard excess liquid and add 30 uL of 50 mM ammonium bicarbonate/5 mM CaCl₂.  Incubate at 37 °C overnight.
   
   
9. The next day, remove the supernatant and keep in a siliconized microcentrifuge tube on ice.
   
   
10. To the gel pieces add 30 uL of 20 mM ammonium bicarbonate, incubate 20 minutes.  Remove the supernatant and pool with the supernatant from previous step.
    
    
11. Add 30 uL of 50% acetonitrile/5% formic acid (made fresh daily, in a glass vial, not plastic), and incubate at RT for 20 minutes.  Remove the supernatant and pool with the supernatant from the two previous steps.
    
    
12. Repeat the previous step (30 uL of 50% acetonitrile/5% formic acid extraction, 20 min incubation) two more times, pooling the supernatants.
    
    
13. Speed-Vac the supernatant to near dryness (e.g. ~20 ul remaining in tube).
     
    
    Cysteine Reduction and Alkylation
    
    1. Rehydrate in 10mM DTT/100mM NH₄HCO₃
    2. Incubate 45 min at 56 C. Remove sample from heat and allow to cool to RT
    3. Aspirate and immediately add freshly made 55mM iodoacetamide/100mM NH4HCO3
    4. Incubate 30 min at RT in dark.
    5. Aspirate
    6. Add acetonitrile till piece turns to sticky white.
    7. Aspirate
    8. Rehydrate in 100 mM NH₄HCO₃ for 5 min
    9. Add equal volume of acetonitrile. Incubate 15 min
    10. Aspirate and dry down in speed vac

Notes:

• Reagent purity is very critical since mass specs can detect low levels of contaminants, including keratins, which are present in old/impure reagents or are present after handing samples/gels without gloves. Therefore, use nanopure water, HPLC grade acetonitrile, high purity formic acid (99%), and if you pour your own gels use new, ultrapure reagents.
  
  
• When making up digestion buffer add CaCl2 to water before adding NH₄HCO₃ to prevent precipitation.

Protocols adapted from:

Shevchenko, Andrej; Wilm, Matthias; Vorm, Ole; Mann, Matthias. Mass spectrometric sequencing of proteins from silver-stained polyacrylaminde gels, Analytical Chemistry, 68: 850-858 (1996).

For Review on gel-separated proteins see:

Patterson, S. D., Reudi Aebersold (1995) Mass spectrometric approaches for the identification of gel-separated proteins. Electrophoresis, 16: 1791-1814.

See other proteolytic digest experimental protocols:
http://pdtc.rockefeller.edu/ms/In-gel%20digstion%20protocol.htm
http://ms-facility.ucsf.edu/ingel.html