One of the ways to remove background on a Western blot

Question: Custom produced antibody is weakly recognizing target protein, even on mitochondrial fraction, where background bands are stronger compared to a target band. What to do in such a case?

Steps of suggested background depleting procedure:

Run the gel electrophoresis and transfer with mutant samples (lacking the target protein) to membrane 1
Prepare the primary antibody solution as for a regular blot
Incubate this solution with the prepared membrane 1
Collect the primary antibody solution and use it on another membrane 2 with the set of samples to be analyzed

Explanation of the procedure:

The antibodies that recognize the background bands will bind to these proteins during incubation, and in this way deplete the antibody preparation of antibodies recognizing unwanted proteins. Depending on protein load/well, 1h/RT or ON/4ºC incubation will be required.

If the target protein is of low expression, located in a cellular compartment like mitochondria, nuclei or chloroplasts, the primary antibody should be antigen affinity purified, which can also help remove background signal as shown here.

Membrane 1 can be re-used following the stripping procedure.

One of the ways to remove background on a Western blot

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