How to improve signal with a weak antibody?

The question to ask in such situations: is the antibody non-specific or is the protocol not optimized?

It is quite common that one and the same protocol, without any modifications is applied for a range of different antibodies.

Each protein-antibody pair may require some protocol modifications in terms of:

Protein load/well
Blotting and membrane type (semi dry or wet, PVDF or nitrocellulose)
Membrane blocking
Primary antibody incubation time 1h/RT or 4°C/ON

As can be seen in the example presented to the right, increasing primary antibody dilution and changing blotting type, contributed to considerable decrease of background signal.

Western blot protocol optimization and interpretation of obtained results, are discussed in details in Agrisera webinars on demand, which can be watched here.

Results obtained before and after optimization of antibody protocol
Protocol adjustment included:

Wet blotting instead of semidry
Blocking in 5 % nonfat milk for 30 minutes longer
Primary antibody used at higher dilution of 1: 4000 ON/4°C instead of 1: 1000 ON/4°C

All protocol details can be found on anti-FERONIA product information sheet.

Aterix marks target proteins: FERONIA.

How to improve signal with a weak antibody?

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