Bio-Rad Immun-Blot® PVDF Membrane - Manual (Page 7)

tocol. Bio-Rad’s Immun-Blot colorimetric blot-
ting kits, or Immun-Star
™ chemiluminescent
kits have excellent blotting protocols.
To minimize non-specific background, the
blocking step and antibody titers are very
important optimization steps. For blocking, we
recommend casein or non-fat dry milk. Typical
concentrations are from 0.2% to 5%. Antibody
dilution factors vary by the manufacture of the
antibody, the specificity, and the purity. To
optimize blot conditions, cut the PVDF into
strips, wet the membrane, and spot the test pro-
tein directly to the membrane. Use different
blocking concentrations and antibody titers to
test the outcomes of these different conditions.
Once the blocking and antibody conditions are
set, you should have good signal to noise results
on the real electrophoretic blots.
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Towbin/SDS buffer:
Towbin buffer as above with addition of up
to 0.1% SDS
Note: Do not add acid or base to adjust pH. The
buffer will range from pH 8.1 to 8.5, depending on the
quality of Tris, glycine, dd H
2
O, and methanol.
Methanol should be analytical reagent grade, as metallic
contaminants in low grade methanol will plate on the
electrodes.
Section 5
Optimization of Blotting
Conditions
Immun-Blot PVDF membrane is a very
versatile blotting membrane. It can be used with
any of the blotting procedures that are used for
immunospecific antigen detection, or total pro-
tein staining. Use the protocol developed for
your lab. If you do not have a standard protocol,
most blotting reagent kits will have a good pro-
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