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

After excising the differentiated spots from the gel, they should be
destained before the in-gel digestion procedure. The destaining of
silver stained gels were as follows:
Following two stock solutions were prepared just prior to use.
Solution A: 2 ml of 100 mM Sodium Thiosulfate: 49.6
mg in 2 ml ultrapure water.
Solution B: 2 ml of 30 mM Potassium Ferricyanide:
19.75 mg in 2 ml ultrapure water.
Solution A and Solution B were combined in a 1:1 ratio to make 4
ml working solution labeled as Reducing Solution.
200 µl Reducing Solution was added to gel tube, and destained
until brownish color has disappeared, approximately 2 – 3 minutes.
Then Reducing Solution was discarded quickly from the tube, and
500 µl ultrapure water was added to wash. Washing step was
repeated 2 times, with 5 minutes each.
Finally, samples were dried in speed vacuum until completely dry,
approximately 15 minutes.
Day Two: Reduction, Alkylation, and Digestion
Step Procedure
1
200 μL of acetonitrile was added and the gel pieces were dehydrated
for ~5 minutes at room temperature. When dehydrated, the gel pieces
were an opaque white color and were smaller in size.
2 Acetonitrile was carefully removed from the sample and discarded.
3
4
The gel pieces were completely dried at ambient temperature in a
vacuum centrifuge for 2-3 minutes.
30 μL of 10 mM DTT was added and the protein was reduced for 30
minutes at room temperature.
5 Then, DTT was carefully removed from the sample and discarded.
6
30 μL of 100 mM iodoacetamide was added and the protein was
alkylated for 30 minutes at room temperature.
cont. on next page
65