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How can I know if I can combine anti Cenp-C and anti Rod1 antibodies? I want to use them to label those protein and analysing cells by fluorescence microscopy.

*Cenp-C is kinetochore protein and Rod1 is localized on the kinetochores or spindle microtubules during cell division.

Can I just combine them if they are from different animals? how can I know they will not have cross reactivity?

Thanks

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  • $\begingroup$ What exactly do you envisage as the potential problem? What do you mean by 'cross reactivity'? $\endgroup$
    – Alan Boyd
    Commented Jul 20, 2017 at 18:24
  • $\begingroup$ I dont know if the the proteins they recognise can be too similar cause they belongs to the same complex so one ab can regonize a similar ag but not the one I want. $\endgroup$
    – Bio
    Commented Jul 21, 2017 at 1:15

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If they are made in different animals you should be fine. You could do a western with those antibodies though, to double-check. Looks like Cenp-C is about 106kD and Rod1 is about 60kD. So on a western, if you see bands at both those sizes with one of the antibodies, you have cross-reactivity between those proteins. And if you just see the specific band you're looking for, you're good.

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  • $\begingroup$ Ok thanks. The fact they need to be from different animals is because the secondary antibody would not be specific? I never studied any course of immunology so I am not sure. $\endgroup$
    – Bio
    Commented Jul 22, 2017 at 3:19
  • $\begingroup$ Secondary antibodies are made to bind to any antibody from a particular animal. If both your primaries are from the same animal, the secondary will bind both of them, and you won't know what signal comes from what protein. If you don't care about distinguishing Cenp-C and Rod1 in your IF, then it doesn't matter. If you do want to be able to distinguish them (ie, have Cenp-C localization red and Rod1 localization green) then the primaries need to be from different animals so you can use two secondaries. $\endgroup$
    – mykinz
    Commented Jul 23, 2017 at 5:15

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