Normally I wash/detect with one primary/secondary-HRP antibody pair, strip, then wash/detect with the other primary/secondary-HRP pair which works well. However, I recently started working with a HRP-conjugated primary antibody for my protein of interest and wondered if I might add this together with the secondary antibody for my loading control to save the stripping and second washing.

Are there any general guidelines for Western blotting with multiple antibodies at the same time, i.e. changing antibody dilution?

An obvious first step is to make sure the HRP-conjugated primary isn't from the animal recognized by the secondary. I anticipate blotting with mouse HRP-conjugated anti-GFP and a secondary rabbit anti-mouse would not be a very good decision.

  • $\begingroup$ Do you mean HRP conjugated secondary? I have not heard of HRP conjugated primary antibodies in the context of Western blotting. $\endgroup$
    – March Ho
    Dec 16, 2014 at 22:06
  • $\begingroup$ I have a HRP-conjugated primary, yes. $\endgroup$
    – Luigi
    Dec 16, 2014 at 23:17
  • 1
    $\begingroup$ it is generally recommended not to use multiple antibodies at the same time mainly due to cross-reactivity/interference either from your primary or secondary Ab since the primary conjugated can bind to the first primary already bound. Also are you confident you can recognise which signal is coming from which Ab? this can be tricky if you have degradation products or dimerisation of your proteins! I generally use one membrane for 1 Ab and another membrane for another Ab. Although this is time consuming but its the safest! $\endgroup$ Dec 17, 2014 at 0:05
  • $\begingroup$ @Bez using a different membrane kind of defeats the purpose of a loading control, doesn't it? Also, in my case there are definitely no multimers or degradation products and the proteins are quite different in size so there should be no confusion as to what is what. $\endgroup$
    – Luigi
    Dec 17, 2014 at 0:41

1 Answer 1


What you can do depends on your proteins: If both proteins (your target protein and the loading control) are seperated far enough, you can detect both of them in the same step by adding both primary antibodies and both secondary ABs into the buffer at the same time. They will bind only to their specific epitope and you will get nice signals. This is something I have done several time. This avoids the stripping process (which can be tricky sometimes) and you have both signals on the same film. Additionally you save chemicals, film and time.

There is another way with which you can come around the stripping: Do you first primary/secondary antibody combination and then incubate the blot with a buffer containing sodium azide. This inhibits the enzyme irreversibly, you have to make to wash very carefully afterwards. Then you can proceed with the second antibody combination.

If you want work with a marked primary antibody (which is unusual, but will of course work) and your loading control at the same time, I would definitely make sure that the secondary antibody with the HRP is not binding to your marked primary, otherwise the signal might get very strong (from the primary and also from the bound secondary), but I don't think it would cause other problems.


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