- Quantitative Proteome 31
- Cut the gel into small pieces with tweezers (about 1 mm in diameter)
and add 200μL of 25 mM TEAB to each sample. Do not remove the
tweezers from the tube until the next step.
- Wash the gel pieces following the below steps:
(1) Add 25 mM TEAB/50% ACN to a final of 1 mL in each sample.
Make sure that no gel pieces remain on the tweezers and remove the
tweezers from the tube.
(2) Vortex the sample for 10 minutes and spin down. Discard the
supernatant and let the level of remaining supernatant be the same
as the level of gel pieces.
(3) Repeat step 1 to step 2 several times until no bubble forms
immediately after vortexing. If the gel pieces shrink greatly, then
proceed to the next step.
(4) Add 25 mM TEAB to a final of 1 mL in each sample.
(5) Vortex the sample for 10 minutes and centrifuge at 13,000 for
30 seconds. Discard the supernatant and let the level of remaining
supernatant be the same as the level of gel pieces. The gel pieces
are harder to precipitate in the 25 mM TEAB than in 25 mM
TEAB/50% ACN.
(6) Repeat steps 1 to 5 until no bubble forms immediately after
vortexing in step 5. It is easier to form bubble when washing with
25 mM TEABC than with 25 mM TEABC/50% ACN.
(7) Dehydrate the gel pieces by adding 100% ACN to a final of 1 mL
in each sample. Vortex erectly until the gel pieces become white
color and spin down. Discard the supernatant and let the level of
remaining supernatant be the same as the level of gel pieces.
(8) Repeat step 7 until the gel pieces aggregate.
- Example of the gel-washing procedures:
Reagent Times
25 mM TEABC/50% ACN 2
25 mM TEABC 1
25 mM TEABC/50% ACN 2
25 mM TEABC 1
25 mM TEABC/50% ACN 1
25 mM TEABC 2
100% ACN 2–3
- Discard all liquid in the tube and leave the aggregated gel pieces only.
- Dry the gel pieces with centrifugal evaporator for about 20 minutes.
