Are blocking conditions nothing to pay attention to?

After protein transfer to PVDF or nitrocellulose membrane, there are still many places on the membrane, which are not occupaid by any proteins, indluding target protein, which is aimed to be detected. Therefore, so called blocking step needs to be included in the protocol, otherwise antibodies will bind to any vacant places on a membrane and this will produce high background signal and false-positive results.

Different blocking reagents can be used to block, like:

Nonfat dry milk 5.10 %
BSA
commercial blockers of proprietary formulation
PVP40 at 0.5-1 % (non protein blocking agent)
Gelatin

Depending upon specific protein-antibody pair, blocking reagent may show different efficiency. Therefore, this step of the Western blot protocol should never be overlooked as properly chosen blocking reagent will help to maximize the signal, especially in case of proteins of low abundance, which are more difficult to visualize.

Take away information for your future blots:

For one and the same protein-antibody pair, different results can be obtained with blocking of PVDF pr nitrocellulose membranes.
Nonfat milk or BSA can not be used as nlockers when working with detection of phosphorylated proteins using phosho-specific antibodies.
Blocking time and temperature 1h/RT or 4ºC/ON, may give different results, depending upon protein-antibody pair.

Blocking is not a trivial step fo Western blot protocol, rather a key factor which is influencing the quality and reliability of a Western blot outcome.

Are blocking conditions nothing to pay attention to?

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