changes in protein profiles in bortezomib applied multiple myeloma ...

7
Day Two: Reduction, Alkylation, and Digestion (cont.)
Then, iodoacetamide was carefully removed from the sample and
discarded.
8 Step 1 was repeated
9 Step 2 was repeated
10
11
The gel pieces were rehydrated with 200 μL of 100 mM ammonium
bicarbonate, incubating the samples for 10 minutes at room
temperature.
Then, ammonium bicarbonate was carefully removed from the sample
and discarded.
12 Step 1 was repeated
13 Step 2 was repeated
14 Step 3 was repeated
15
16
17
18
19
20
21
22
23
30 μL of the trypsin solution was added to the sample and the gel
pieces were allowed to rehydrate on ice for 10 minutes with
occasional vortex mixing.
The gel pieces were driven to the bottom of the tube by centrifuging
the sample for 30 seconds.
5 μL of 50 mM ammonium bicarbonate was added to the sample and
the mixture was vortexed.
The sample was driven to the bottom of the tube by centrifuging the
sample for 30 seconds. Digestion was carried out overnight at 37ºC.
Day Three: Extraction of Peptides for Analysis
30 μL of 50 mM ammonium bicarbonate was added to the digest and
the sample was incubated for 10 minutes with occasional gentle vortex
mixing.
The digest was driven to the bottom of the tube by centrifuging the
sample for 30 seconds.
The supernatant was carefully collected and the sample was
transferred to a 0.5 ml plastic micro centrifuge tube.
30 μL of extraction buffer was added to the tube containing the gel
pieces and incubated for 10 minutes with occasional gentle vortex
mixing.
The extract was driven to the bottom of the tube by centrifuging the
sample for 30 seconds.
cont. on next page
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