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

electrophoresis, water circulation was used and electrophoresis was started at constant
current at 32 mA for 30 minutes and followed by 50 mA for 5 – 6 hours. To monitor the
progress of the electrophoresis, Bromophenol Blue was used. When it reached the
bottom of the gel, electrophoresis was stopped.
3.2.9.3. Staining of the Gel
A number of staining techniques have been developed to stain polypeptides with
silver salts after separation by two-dimensional gel electrophoresis. In every case, the
procedure based on differential reduction of silver ions that are bound to the side chains
of amino acids (Switzer et al., 1979; Nielsen and Brown, 1984; Merril et al., 1984).
1
Table 3.7. Time Coarse for MALDI-TOF Compatible Mass Spectrometry
(Source: Adapted from He et al., 2003; He et al., 2004; Lin et al., 2008)
STEP SOLUTION
Fixing
(Fixer Soln.)
40% EtOH,
10% HAc;
TIME OF
TREATMENT
> 1 hour
(overnight)
COMMENTS
Overnight incubation is
all right
2 Washing -
Pretreatment Soln.
- -
3 Sensitizing 30% EtOH,
4.1% NaAc,
0.2% Na2S2O3;
30 minutes Time should be exact
4 Washing dH2O 5 min. x 3 2 nd & 3 rd Silver Nitrate Solution
solution should
always be fresh
5 Impregnate 0.1% AgNO3, 0.02%
formalin;
20 min.
-
6
7
Washing
Developing
8 Stop
dH2O
Developing Solution
2.5% Na2CO3, 0.01%
formalin
Stop Solution
1.46% EDTA
1 min. x 2
~ 10 min.
> 10 minutes
9 Washing dH2O 5 min. x 3
Time should be exact
2 nd & 3 rd solution should
always be fresh
After a few minutes, add
some dH2O to proceed
the reaction slowly. Time
should be determined by
observation of color
development
The gels can be kept in
this solution overnight
2 nd & 3 rd solution should
always be fresh
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