Appendix a: technical notes – Hoefer PR625 Immunoblot User Manual
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Appendix A: Technical notes
Note 1: Blocking the membrane
Membranes should always be blocked before
mounting in the ImmunoBlot. Please follow
instructions for blocking provided in your
Western blotting detection kit.
Generally, 5% Non-fat dry milk or 5% BSA are
used as blocking agents.
Blocking with Tween-20 or other detergents
alone is not recommended as it may cause slow
lateral diffusion of proteins through the nitrocel-
lulose membrane. We recommend performing
the initial blocking step in a solution containing
protein (such as 5% BSA) without detergent.
Avoid extensive washes with solutions contain-
ing no protein prior to mounting the membrane
in the ImmunoBlot.
In many cases, satisfactory blocking may be
attained by incubation with PBS/10% newborn
calf serum/0.1% Tween-20 for a minimum of
one hour at room temperature. However, block-
ing agents differ in their efficacy depending upon
circumstances. For blots of whole cell lysates,
blocking with 50 to 100% serum can be very
effective in reducing non-specific binding.