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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E.<br />
F.<br />
G.<br />
STOCK REAGENT PREPARATION (cont.)<br />
10% Ammonium Persulfade (w/v)<br />
Dissolve 100 mg ammonium persulfate <strong>in</strong> 1 ml distilled water<br />
Sample Buffer (SDS reduc<strong>in</strong>g buffer: 62.5 Mm Tris- HCl, pH 6.8, 20% Glycerol, 2%<br />
SDS, 5% β- Mercaptoethanol)<br />
Distilled water 3.0 ml<br />
0.5 M Tris- HCl, pH 6.8 1.0 ml<br />
Glycerol 1.6 ml<br />
10% SDS 1.6 ml<br />
β- Mercaptoethanol 0.4 ml<br />
0.5% (w/v) bromophenol blue (<strong>in</strong> water) 0.4 ml<br />
Total 8.0 ml<br />
Dillute the sample at least 1:4 with sample buffer. Heat at 95°C for 4 m<strong>in</strong>utes.<br />
5x Runn<strong>in</strong>g Buffer (1x= 25 mM Tris, 192 mM glyc<strong>in</strong>e, 0.1% SDS, pH 8.3)<br />
Tris base 45.0 g<br />
Glyc<strong>in</strong>e 216.0 g<br />
SDS 15.0 g<br />
Distilled water to 3 L. Do not adjust the pH with acid or base. Store at 4°C.<br />
Warm to 37°C before use if precipitation occurs. Dillute 300 ml 5x stock with<br />
1.2 L distilled water for one electrophoretic run.<br />
To prepare 100 ml of 1X TGS buffer, 20 ml of 5X TGS buffer was diluted to a<br />
f<strong>in</strong>al volume of 100 ml with ultra pure water.<br />
B.6.2.2. Preparation of Overlay Agarose<br />
SDS Electrophoresis Buffer<br />
Tris Base 3.03 g<br />
Glyc<strong>in</strong>e 14.40 g<br />
SDS 1.0 g<br />
were dissolved <strong>in</strong> 1000 ml of distilled water<br />
Overlay Agarose<br />
Agarose 0.50 g<br />
Bromophenol Blue 2.0 mg<br />
SDS Electrophoresis Buffer 100 ml<br />
stored at 4°C<br />
110