Hello Ahmed Al-Omar

It is fine to mix several antibodies and incubate them together on the same membrane only if the below conditions are met.

1. The primary antibodies you are using are of good quality and you have achieved a good separation of your protein of interest namely, protein bands are very distinct (no unspecific bands) and very well size separated.

2. Ensure that your primary antibodies have little background (test them individually, if necessary) so as to not to mistake the background for any signal.

3. When performing a multiplex western blot, use primary antibodies from different host species for each target being probed, which you have already planned.

4. Also, use a combination of antibodies from two distantly related species such as rat and rabbit, avoiding combinations like mouse and rat or goat and sheep. This will aid in the selection of appropriate secondary antibodies to minimize potential antibody cross-reactivity, which may lead to confusing results.

So, for your primary antibody in mouse host species, use rabbit as the secondary antibody host species.

For primary antibody in rabbit host species use mouse as the secondary antibody host species.

For primary antibody in goat host species, use rabbit or mouse as the secondary antibody host species.

Best.

Hi Ahmed Al-Omar

In addition to was mentioned, I would like to aware you about two important observations while using several P.Abs at once:

1. Larger membrane may need larger amount of P.Ab solution and as a result larger volume of your precious antibody.

2. If you use ECL, the presence of too much bands may affect the intensity of the light.

Best .....

Dear Ahmed Al-Omar

I concur with Malcolm Nobre's answer and his recommendations listed in points 1-4.

Generally, it is important to know how well every single antibody works. So, individual antibody establishments are crucial to find optimal incubation and detection conditions. You want be 100% sure that the band you detect is the protein of interest, and that there are no overlaps with the other proteins you are analyzing.

Moreover, it is important to establish antibody concentrations for achieving comparable signal intensities when multiplexing, as you do not want the signals to be at the extremes of your detection system's dynamic range. Strong signals should also not cover faint bands. Ideally, you have access to a fluorescence-based imaging system which allows you multichannel detection. Thus, you may distribute your different antibodies among the provided fluorescence channels to avoid signal overlaps or re-detection. Also, keep in mind - when combining primary antibodies from three different species - that the utilized secondary antibody must not originate from the same host species as one of the used primary antibodies. E.g., when using a primary antibody made in goat, and you consequently need to apply an anti-goat secondary, do not use other secondary antibodies made in goat, as the anti-goat secondary will detect those ones too.

Finally, when multiplexing is not possible, cutting membranes or re-probing them after performing a stripping protocol can constitute alternative approaches.