32 A Practical Guide to Cancer Systems Biology
- Prepare the trypsin solution:
(1) If you will exhaust the whole bottle containing 20μg trypsin, then
add 200μL of 25 mM TEAB to dissolve trypsin powder.
(2) If you will use a part of the bottle of trypsin, then add 25μLofthe
trypsin resuspension dilution buffer supplied in the kit to dissolve
trypsin powder.
- The reagent for dissolving trypsin must be precooled at 4◦C.
- Add appropriate amount of trypin to the dehydrated gel pieces and
allow to stand on ice for a while to let the gel pieces absorb the trypsin
solution. The amount of protein chosen initially to the amount of trypsin
is 10:1 (g/g). - Add 25 mM TEAB to rehydrate the gel pieces. The volume of 25 mM
TEAB is 2.5 times the volume of the gel pieces, which is determined
by the initial volume of sample and the volume of acrylamide/bis-
acrylamide, APS and TEMED added previously. - Let the sample stand on ice for 10 minutes and observe the level of
25 mM TEAB in the tube. The level of 25 mM TEAB must be about
5 mm higher than the level of gel pieces. - If the amount of 25 mM TEAB is not enough, then add additional
amount of 25 mM TEAB to let the reagent cover the gel pieces. Let
the sample stand for another 10 minutes and observe the level of 25 mM
TEAB again. - Repeat step 9 to make sure the 25 mM TEAB is enough to cover the gel
pieces. - Cap and seal the 2 mL protein lobind tube containing sample with
parafilm carefully. - Incubate the tube in a water bath at 37◦C overnight (16–20 hours).
Gel extraction to obtain peptides^17
- Prepare several new 2 mL protein lobind tube as final peptide tubes.
Prepare several new 1.5 mL centrifuge tubes, some as mixture (of gel
pieces and peptides) collection tubes and some as peptide collection
tubes. - Spin down the sample from the water bath. Transfer the supernatant to
the mixture collection tube and let the level of remaining supernatant
be the same as the level of gel pieces. - Add 200μL of 25 mM TEAB to the gel pieces.