changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
changes in protein profiles in bortezomib applied multiple myeloma ...
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After excis<strong>in</strong>g the differentiated spots from the gel, they should be<br />
desta<strong>in</strong>ed before the <strong>in</strong>-gel digestion procedure. The desta<strong>in</strong><strong>in</strong>g of<br />
silver sta<strong>in</strong>ed gels were as follows:<br />
Follow<strong>in</strong>g two stock solutions were prepared just prior to use.<br />
Solution A: 2 ml of 100 mM Sodium Thiosulfate: 49.6<br />
mg <strong>in</strong> 2 ml ultrapure water.<br />
Solution B: 2 ml of 30 mM Potassium Ferricyanide:<br />
19.75 mg <strong>in</strong> 2 ml ultrapure water.<br />
Solution A and Solution B were comb<strong>in</strong>ed <strong>in</strong> a 1:1 ratio to make 4<br />
ml work<strong>in</strong>g solution labeled as Reduc<strong>in</strong>g Solution.<br />
200 µl Reduc<strong>in</strong>g Solution was added to gel tube, and desta<strong>in</strong>ed<br />
until brownish color has disappeared, approximately 2 – 3 m<strong>in</strong>utes.<br />
Then Reduc<strong>in</strong>g Solution was discarded quickly from the tube, and<br />
500 µl ultrapure water was added to wash. Wash<strong>in</strong>g step was<br />
repeated 2 times, with 5 m<strong>in</strong>utes each.<br />
F<strong>in</strong>ally, samples were dried <strong>in</strong> speed vacuum until completely dry,<br />
approximately 15 m<strong>in</strong>utes.<br />
Day Two: Reduction, Alkylation, and Digestion<br />
Step Procedure<br />
1<br />
200 μL of acetonitrile was added and the gel pieces were dehydrated<br />
for ~5 m<strong>in</strong>utes at room temperature. When dehydrated, the gel pieces<br />
were an opaque white color and were smaller <strong>in</strong> size.<br />
2 Acetonitrile was carefully removed from the sample and discarded.<br />
3<br />
4<br />
The gel pieces were completely dried at ambient temperature <strong>in</strong> a<br />
vacuum centrifuge for 2-3 m<strong>in</strong>utes.<br />
30 μL of 10 mM DTT was added and the prote<strong>in</strong> was reduced for 30<br />
m<strong>in</strong>utes at room temperature.<br />
5 Then, DTT was carefully removed from the sample and discarded.<br />
6<br />
30 μL of 100 mM iodoacetamide was added and the prote<strong>in</strong> was<br />
alkylated for 30 m<strong>in</strong>utes at room temperature.<br />
cont. on next page<br />
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