I have done IFA (immunofluorescence assay) previously but i targeted only one protein or i tagged my endogenous protein with GFP/RFP then i performed IFA. I want to do the IFA in parental cell line (HEK cells). My aim is to show the two proteins are co-localized or not?

  1. Protein-1 (secondary antibody Mouse)
  2. Protein-2 (secondary antibody Rabbit)

How can i co-localize two different proteins in the same sample? Which primary antibody do i have to add first? can any one please suggest me something?


1 Answer 1


If you search the web or Google Scholar, you should be able to find plenty of examples of colocalization protocols.

In general, though, for each target you're going to want to have an unstained control sample (you can use one for all targets), a secondary-only sample to look for non-specific background staining, and a single-color sample (primary plus secondary). Then, you'll have a sample with both primaries and both secondaries. You always add the primaries at the same time, and the secondaries at the same time.

As you start getting into higher numbers of colors, you'll also need an FMO (fluorescence minus one) control for each color so you can test for signal bleed-over from one channel to another. This sample will combine all the colors except for one.

  • $\begingroup$ Thanks for your answer. It is really helpful. one more important question i have. Is there any way to do the RNAase treatment in IFA? I suspect if my protein of interest co-localization RNA based? $\endgroup$
    – Rengaraj
    May 23, 2021 at 9:03
  • 1
    $\begingroup$ @Rengaraj yes, it is possible, although I haven't done it myself. Here is a ResearchGate discussion (with references) on how to do it. $\endgroup$
    – MattDMo
    May 23, 2021 at 18:47
  • $\begingroup$ @Rengaraj, also, if this answer addressed your problem, please consider accepting it by clicking on the check mark/tick to the left of the answer, turning it green. This marks the question as resolved to your satisfaction, and awards reputation both to you and the person who answered. Since you have >= 15 reputation points, you may also upvote the answer if you wish. There is absolutely no obligation to do either. $\endgroup$
    – MattDMo
    May 23, 2021 at 18:49

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