Biology Reference
In-Depth Information
population. Ammonium sulfate precipitation is performed as
follows: dialyze 100 mL of the extracellular medium with
50 mM sodium acetate buffer pH 5.0 overnight at 4 °C to
remove salts remaining in the culture medium. Add 65 g
(NH
4
)
2
SO
4
(up to 95 % saturation) to 100 mL of extracellular
medium and maintain for 1 h at 4 °C on the stirrer. The percent
of ammonium saturation should be adjusted according to pre-
cipitate a particular protein fraction [
46
]. Centrifuge at
3,500 ×
g
for 20 min at 4 °C. Discard the supernatant, and
resuspend the precipitated protein in 50 mM sodium acetate
buffer pH 5.0. A last step for dialyzing protein sample should
be introduced in order to remove salts which will interfere
with isoelectrofocusing (IEF). Dialysis is carried out overnight
at 4 °C in 50 mM sodium acetate buffer pH 5.0.
17. TCA is a very effective precipitant compound. TCA-based pre-
cipitation is a popular method for sample preparation of both
one- and two-dimensional gel electrophoresis, because it can
concentrate samples, remove salts and polysaccharides, and dena-
ture endogenous proteases. Limitations of TCA precipitation
include diffi culties in the protein resolubilization that can be
solved by the aid of a sonicator probe. Special care should be paid
to wash protein pellets with acetone or methanol to remove
residual TCA prior to electrophoretic analysis or mass spectro-
metric analysis due to the potential protein degradation or modi-
fi cation under extended exposure to this low pH solution.
18. Do not overdry protein precipitate in order to facilitate its resol-
ubilization. Protein precipitate is resuspended either in one- and
two-dimensional electrophoresis sample buffer if the following
analysis is performed by one- or two-dimensional electropho-
retic analysis, respectively. For one-dimensional electrophoresis,
resuspend the protein precipitate in lysis buffer (7 M urea, 2 M
thiourea, 30 mM Tris-HCl, pH 9.0, and 4 % CHAPS). Store at
4 °C, and for two-dimensional electrophoresis use rehydration
solution with IPG buffer (Subheading
2.8
).
19. To avoid air bubble entrapment, insert the well-forming comb
inclined at one edge.
20. Check the electrical connections on the cell to ensure that
solution is not in contact with either banana plug, and connect
the anode to the (+) terminal on the power supply, and the
cathode to the negative terminal.
21. Preclude excessive heating by placing the chamber in a cold
room.
22. For staining proteins a highly sensitive silver staining technique
can be used which permits detection of polypeptides in
polyacrylamide gels at concentrations 100-fold lower than
Coomassie brilliant blue R-250.
