MANUAL TRYPSIN IN-GEL DIGESTION PROTOCOL FOR COOMASSIE STAINED GELS
Coommassie de-stain gels: by washing briefly with 200µL NH4HCO3 (Solution A) to make sure gel pieces are at the correct pH.
Add 200µL 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) into each well. Vortex to mix and incubate for 10 minutes. Remove liquid.
Repeat step 2. Gel pieces should be clear at this stage. If they are still blue, repeat as necessary until color is gone.
Wash for 5 min with 50µL of 100% Acetonitrile (Solution C) to dehydrate gel pieces. Vortex during incubation.
Remove Acetonitrile, then let air-dry for 10 min. The gel pieces should be noticeably shrunken and probably white.
Reduction and Alkylation: Cover gel pieces with 50µL 10mM DTT in 50mM NH4HCO3 (Solution D). Let proteins reduced for 45-60 min in 37oC incubator.
Remove DTT solution and add 50µL of 55mM iodoacetamide in 50mM NH4HCO3 (Solution E). Incubate for 45 min in dark place at room temperature.
Remove iodoacetamide, discard.
Wash gel pieces with 200ul of NH4HCO3 (Solution A) for 5 min with vortexing. before adding 100ul of 100% Acetonitrile (Solution C).
Remove liquid after 5 min, discard.
Wash gel pieces with 50ul 100mM NH4HCO3 (Solution A) for 10 min, then twice with 200ul 50% Acetonitrile / 50% 100mM NH4HCO3 (Solution B) for 10min.
Dehydrate with 100 ul 100% Acetonitrile (Solution C) for 5 min as above.
Remove remaining liquid and let the gel dried.
Trypsin Digestion: Prepare trypsin mix to final concentration of 13ng/µL in 50mM Ammonium bicarbonate.
Add 30µL (or more if required) of mixed trypsin solution to cover the gel pieces.
Allow 30 min for gel rehydration at 4oC (on ice).
Digest overnight at 37oC.
Peptide Extraction: Transfer the digest solution supernatant (if any) into clean 0.65ml Eppendorf tubes.
To the gel pieces, add 50 µL of 50% acetonitrile / 2% formic acid, incubate 30 min. Spin, remove supernatant and combine with initial digest solution supernatant. Please note that total volume may vary depending on the gel sizes.
Vortex the extracted digests, speed vac to reduce volume to 10 µL. If the remaining volume is less than 10ul, use 2% formic acid to bring the volume up to 10 µL.
Spin at 14,000 rpm for 30 min to remove any micro particulates.
Transfer the supernatant to a fresh 0.65ml eppendorf tube for storage at 4oC fridge OR directly into PCR plate for Mass spec for analysis.
Reagents
Buffer 100mM Ammonium Bicarbonate
ADD 0.78 g ammonium bicarbonate to 100ml ddH20. Fill first medium size reservoir.
Reduction: - Dithiothreitol (DTT)
Add 15 mL of 100 mM ammonium bicarbonate to 23.1 mg of DTT to give a solution of 10 mM DTT.
Alkylation - Iodoacetamide(DTT)
Add 15 mL of 100 mM ammonium bicarbonate to 153 mg of iodoacetamide to give a solution of 55 mM iodoacetamide - 15 mL of each is enough for up to 96 samples.
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