Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 170

11. Not only the Sample Buffer provides the denaturing environment
for the proteins, but the bromophenol blue also provides the
dye front to indicate how far along the resolved proteins have
travelled to. So, when the bromophenol blue has reached
the end of the polyacrylamide gel, the voltage is stopped.
12. The gel is then removed from the Protean II system and placed
onto a piece of blotting paper prewet with Transfer Buffer.
A piece of prewet nitrocellulose or PDVF membrane is placed
on top of the gel followed by another piece of prewet blotting
paper. This sandwich is further fl anked by two pieces of fi ber
pads before placing it into the transfer cassette of the Bio-Rad
Transfer System. Complete anatomy of the Western Transfer
sandwich is shown in Fig. 3 .
13. The transfer cassette is inserted into the electrode assembly
before placing it into the transfer tank. Attach the electrodes
and set the power supply to 50 mA at constant current to
transfer over night. Alternatively the power supply can be set
to 100 V at constant voltage to transfer for 1 h. Please consult
the Instruction Manual when other systems are used.
Part 2
1. Upon completion of the transfer, the membrane is removed
from the assembly and is placed into the Blocking Buffer for at
least 1 h.
2. Dilute the primary antibody in 3% nonfat milk solution in TBST
and incubate at room temperature for at least 2 h. Nevertheless,
the optimum duration and dilution factor is dependent on
the potency and effi cacy of the antibody. If primary antibody
Fig. 3. The anatomy of a Western Transfer sandwich.