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

B.6.2.3. Preparation of In-Gel Digestion Chemicals
Ammonium Bicarbonate (100 mM) : 0.2 g of ammonium bicarbonate was
dissolved in 20 ml of water.
Ammonium Bicarbonate (50 mM) : 2 ml of 100 mM ammonium
bicarbonate was mixed with 2 ml of water.
DTT (10 mM) : 1.5 mg of dithiothreitol was placed in a 1.5 ml plastic
centrifuge tube followed by addition of 1 ml of 100 mM ammonium
bicarbonate for complete dissolving of DTT.
Iodoacetamide (100 mM) : 18 mg of iodoacetamide was placed in a 1.5 ml
plastic centrifuge tube followed by addition of 1 ml of 100 mM
ammonium bicarbonate for complete dissolving of iodoacetamide.
Trypsin Solution : 1 ml of ice cold 50 mM ammonium bicarbonate was
added to 20 μg of sequencing-grade modified trypsin (V5111; Promega)
and dissolved by drawing the solution into and out of the pipette. The
trypsin solution was kept on ice until use. The final concentration is 20
ng/ml trypsin.
Extraction Buffer : 10 ml of acetonitrile (Merck) was added to 5 ml of
water followed by addition of 1 ml of formic acid (Merck) and the final
volume was adjusted to 20 ml with water. The final concentrations were
50 % (v/v) acetonitrile and 5 % (v/v) formic acid.
B.6.2.4. Preparation of MALDI Matrix
α-cyano-4-hydroxycinnamic acid (HCCA) was used as a matrix in proteomics
studies and it was prepared with two layer method.
First Layer : Approximately 3–6 mg of HCCA was weighted in 100 µl methanol
(MeOH). Then 400 µl acetone was added to the mixture and dissolved all of them.
Increase MeOH part on very dry days and lower it on very humid days.
Second Layer : Approximately 5 mg of HCCA was weighted into a plastic vial
and 200 µl MeOH was added. Firstly, all the matrices into MeOH were dissolved and
than, 300 µl deionized water which contains 0.1% TFA was added. HCCA started to
precipitate out. Finally, it was centrifugated at maximum speed for nearly 8 minutes.
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