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Question continued: I was doing a triple staining(staining 3 proteins) with a different combination of antibodies from different species and was irritated by choices. Should  I choose goat anti-rabbit conjugated to Alexa488 or donkey anti rabbit conjugated to Cy3 and so on…Is there some tool where we can input our constrains  and the tool presents us with possible options ?

Tirumalai Kamala’s answer: Antibody choice is one of most important decisions a researcher makes in their research. Never to be undertaken lightly and yet it has been for far too long. And that’s why it’s one of the major reasons for the current reproducibility crisis in biomedical science (1).

Not one, not two, but three immunofluorescence stains? Quite complicated. You’d need a checker-board of controls necessary to validate each stain within the panel, especially so if all three stains involve secondary antibodies. Controls become even more necessary because of the much higher chance of generating artifacts with a triple stain. Also need to account for other variables: sample species, suspension cultures/tissues, fresh/fixed, target antigens abundant/sparse, primary antibodies monoclonal/polyclonal. I surmise your primary antibody’s rabbit but rabbit what? IgM? IgG?

I’d also be asking some yes/no questions:

  • Am I choosing products referenced in peer-reviewed publications?
  • Have the antibodies been independently validated for immunofluorescence?
  • Are there reported cross-reactivities?
  • Have cross-reactivities been minimized/mitigated by absorption?
  • Does fluorophore conjugation affect antibody binding to its antigen? If yes, what’s a suitable fluorophore for this antibody for immunofluorescence?


Antibodies-online would be one place I’d use to select between options. Primary Antibodies | antibodies-online.com

So for example, if you search antibodies-online for Cy3 conjugated donkey anti-rabbit, not even a single product shows up and if you do the search for Cy3 conjugated goat anti-rabbit, seven products show up, none of which are reported to be independently validated for immunofluorescence, and only one of which is associated with 6 publications cited on PubMed.

Once I had a preliminary list of possible antibody candidates I planned to use, I’d run them through additional antibody resources to get as much information about them as possible. This would further help me rule out dodgy reagents. Those other antibody resources:
Antibody encyclopedia: Explore Antibodypedia
Universal Identities for Antibodies: Antibody Registry
Independent Antibody Reviews: pAbmAbs antibody blog and review site

I think you should consult an expert for step-by-step guidance, and I can’t think of anyone better qualified than David L. Rimm at the Department of Pathology, Yale University School of Medicine, New Haven, Connecticut, USA. He has developed the following multistep flowchart for appropriate validation of antibodies for IHC (ImmunoHistoChemistry) and QIF (Quantitative ImmunoFluorescence) (2). All his advice isn’t eminently practical. For e.g., performing knockdown of target antigens using siRNA to validate each and every target is unlikely to be every researcher’s cup of tea. Too expensive and time-consuming. Nevertheless, his is a very useful decision-making chart that’ll improve the validity of the data you generate. You could also email him directly (david.rimm@yale.edu) for advice.

 


ELISA and flow cytometry are more my forte but I’ll be able to give pointers on best practices. If you need more help, just message me directly.

I cannot urge you strongly enough to read both references in their entirety.

Bibliography

  1. Baker, M. “Reproducibility crisis: Blame it on the antibodies.” Nature 521.7552 (2015): 274. Page on nature.com
  2. Bordeaux, Jennifer, et al. “Antibody validation.” Biotechniques 48.3 (2010): 197. Page on biotechniques.com

 

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