Starting with 1mm x 1mm gel piece:
1. Wash gel plug in 100 ul solution B for 20 min (to equilibrate to pH 8ish and to remove the coomassie blue).
2. Repeat step 1.
3. Wash briefly with 100 ul acetonitrile to remove aqueous solutions.
4. Shrink plug by washing in 100 ul acetonitrile for 10 to 15 min.
5. Air dry for 10 min to remove all of the acetonitrile.
6. Add DTT soln: 100ul of 10mM DDT in 50mM Ammonium bicarbonate - 30 min @ 60C in heating block.
7. Remove liquid (do not shrink gel)
8. Add Iodoacetamide soln: 100ul of 100mM in 50mM Ammonium bicarbonate - 30 min @ room temp in dark. Use a brown (UV resistant) eppendorf.
9. Remove liquid.
10. Repeat step 1.
11. Repeat step 1. (The sample can be frozen at this point if necessary).
12. Wash briefly with 100 ul acetonitrile.
13. Shrink plug by washing in 100 ul acetonitrile 10-15 min
14. Remove all acetonitrile and Air dry for 10 min
15. Add trypsin soln: 5 ul containing 50 ng in 10 mM ammonium bicarbonate (Promega modified porcine trypsin)
16. Incubate in 37 degree water bath for 3 hr with tube lids on (2-4 h works OK)
17. Do not recover condensation on lid. Add 5 ul 5% formic acid, agitate by tapping tube, stand for 10 min.
18. Flash freeze in liquid nitrogen & store in minus 80 freezer until needed.
19. Defrost. Sonicate in ultrasonic bath for 10 mins (this is optional). Spot 0.25 - 0.5 ul on MALDI target. Wash spot with 3 ul 5% formic when dry.

Solution B is freshly prepared 400 mM ammonium bicarbonate : 100% acetonitrile in a ratio of 1:1

Alternatively: 0.1 mM TCEP in 50 mM ammonium bicarbonate can be used inplace of DTT in step 6.

DDT: 154 mw => 2.31 mg/ 1.5 ml.
Iodoacetamide: 185 mw => 27.75 mg/ 1.5 ml.

Trypsin: add 100 ul of promega re-suspension buffer to one vial of promega trypsin. Aliquot as 5 ul, flash freeze in liquid nitrogen and store at -20C. To one aliquot add 95 ul of 10 mM ammonium bicarbonate and use 5 ul per digest.