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In Gel Digestion of Proteins Separated by PAGE

In Gel Digestion of Proteins Separated by PAGE (According to EMBL Protein and Peptide Group, 1997)


Extracted from: http://www.pharma.ethz.ch/bmm/protocols/ingeldig.html, with some modifications.


  1. Excision of protein band or spots from polyacrylamide gels:
     
    • Rinse your gloves with water to avoid traces of dust in the sample.
    • Rinse the gel with water.
    • Excise spots with a clean pipette tip cut off (to the correct diameter) or with a razor blade, cutting as close to the edge of the spot as possible (get as little background gel as possible). Cut into small particles.
    • Transfer gel into an Eppendorf tube.
       

  2. Reduction and Alkylation:

    • Wash the cut-up particles with 100-150 ul of water (5 min), spin down (table-top centrifuge, max. 2800 rpm) and remove liquid.
    • Add acetonitrile (3-4 gel volumes) and wait 10-15 min, spin particles down and remove all liquid, dry down in a speed vac.
    • Swell the gel pieces in 10 mM DTT/0.1 M NH4HCO3, enough to cover the gel, and incubate for 30 min at 56ºC (water bath) to reduce the protein.
    • Spin down and remove excess liquid, shrink with acetonitrile.
    • Replace acetonitrile with 55 mM Iodoacetamide/0.1 M NH4HCO3, incubate 20 min at room T in the dark.
    • Remove iodoacetamide solution, wash the gel particles with 150-200 ul 0.1M NH4HCO3 for 15 min.
    • Spin down, remove all liquid and shrink with acetonitrile.
    • Remove all liquid and dry gel particles down in speed vac.


  3. Washing Gel Pieces to remove unpolymerized acrylamide and contaminants:

    • Wash the sample 5 min in 80 ul 0.1 M NH4HCO3
    • Add an equal volume of acetonitrile and vortex 5 min.
    • Spin down the particles and remove the solution.
    • Repeat this wash step twice.
    • Shrink the gel pieces with acetonitrile about 5 min
    • Remove the solution and dry down in a speed vac.


  4. In-gel digestion with trypsin:

    • Pre-cool tubes with the dried gel particles and the trypsin on ice.
    • Rehydrate the gel particles in the trypsin solution (20 ng/ul Trypsin(Promega #V5111) in 25 mM NH4HCO3) for 30-45-min at 4ºC. Add just enough solution to cover (~20 ul) the gel pieces.
    • After 15 min, check the sample and add more trypsin solution if the solution has been completely absorbed by the gel pieces.
    • After a total of 30-45 min rehydration, remove the remaining trypsin solution.
    • Add 10-75 ul (to just cover) 25 mM NH4HCO3 without trypsin and incubate 16-20 hours at 37ºC.
    • Spin down the condensed water droplets.
    • Transfer liquid supernatant which can already contain small peptides (<2000 Da) to a fresh 0.5 tube. Keep on ice.


  5. Extraction of Peptides from Gel:

    • Add 10-15 ul of 25 mM NH4HCO3.
    • Incubate 15 min at 37ºC while shaking (vortex every 5 during incubation)
    • Spin down and add acetonitrile (1-2 times the volume of the gel ~10-80 ul).
    • Incubate 15 min at 37ºC while shaking.
    • Sonicate for 5 min at 37ºC.
    • Spin down liquid and transfer the peptide containing solution to the same tube saved before containing peptides.
    • Add 10-50 ul of 5% formic acid (to adjust pH).
    • Incubate 15 min at 37ºC while shaking.
    • Spin down and add acetonitrile (1-2 times the volume of the gel).
    • Incubate 15 min at 37ºC while shaking.
    • Sonicate for 5 min at 37ºC.
    • Spin down and transfer the peptide containing solution to the same tube as before, with basic extracted peptides.
    • Dry in a speed vac.