In-Gel Digestion Proteomics Protocol for Peptide Mass Fingerprinting and Mass Spectrometry

0.1 M Ammonium Bicarbonate be made fresh for each set of digestion (can be stored for one day in the refrigerator, do not need a filter)

washing
1. Place water into the tube maxymum recovery.
2. Excise piece band Coomassie stained using P1000 pipette tip to the bottom punching, and then sucked with the pipette.
3. Can repeat for a few pieces before the pieces into the tube pipeting maxymum recovery.
4. Wash the gel pieces by placing 100uL of 0.1 M ammonium bicarbonate and sonicating for 15 minutes (no heat).
5. Turn down the liquid.
6. Add the same volume (100uL) of acetonitrile and sonicate for 15 minutes
7. spin down liquids.
8. Remove all liquids and repeat wash steps if needed
9. After each washing step, place the tubes on the sheet of white paper to check if all the pieces are white gel
10. If they are white, do not need to do anymore laundering.
11. Add acetonitrile and shrink.

In-Gel Digestion Proteomics Protocol for Peptide Mass Fingerprinting and Mass Spectrometry

Reduction and Alkylation
1. Add 50uL10mM DTT in 0.1 M ammonium bicarbonate 56 degrees C, 30 minutes, shaking.
2. spin down, remove, and add acetonitrile to shrink.
3. Add 50uL 55mm Iodoacetamide in 0.1 M ammonium bicarbonate and 20 minutes incubate in the dark; The movie every now and then.
4. Turn down.
5. Wash with 0.1 M ammonium bicarbonate (150-200uL). Repeat if desired.
6. spin down.
7. Shrink with acetonitrile.

Digestion
1. rehydrate in digestion buffer.
The composition of the digestion buffer: water 50uL, 50uL 0.1 M ammonium bicarbonate, 15uL 0.1ug / uL pork trypsin, 5uL 1% calcium chloride)
2. Turn down and remove after 30 minutes on ice.
3. Add more of the trypsin digestion buffer without.
4. Incubation overnight was 37 degrees C with shaking.

repeal
1. The next day, transfer the digestive tube new maxymum recovery.
2. Add 20uL 0.1 M ammonium bicarbonate and sonicate for 15 minutes
3. Turn down.
4. Add 40uL acetonitrile and sonciate for 15 minutes.
5. Remove and incorporate into the digestive tube.
6. Add 50uL of formic acid and sonicate for 15 minutes.
7. Add 100uL acetonitrile and sonicate for 15 minutes.
8. Combine.
9. Divide into two or more tubes if necessary, freeze in liquid nitrogen, lyophilize and freezes at 20 degrees C.