20% of Antibodies not specific?

gravenewworld

Nice little paper about properly validating your reagents:

http://www.ncbi.nlm.nih.gov/pubmed/22361696


Why is it in academia absolutely no one validates their reagents like antibodies while in industry it is absolutely required?

Rather than admitting their results are due to nonspecific binding, I've seen some papers where academics attribute their results to say something like another unknown isoform of the protein that they're studying.

Why is it that the peer review system has completely and utterly failed to to address this type of terrible science? What's worse is that once results that come from an antibody that might not be specific is published, many other labs go out and start using the same antibody to study the protein(s) in question.

What a complete an utter joke a lot of science being done now has become. Manufacturers are notorious for being lazy when it comes to validating their antibodies, academics simply take it for granted that they are supposed to work. Maybe it is time our universities do high quality science with validation like industry?

Monique

I always validate my antibodies and indeed I've found many commercial antibodies to be non-specific. I don't recognize your statement that in academia no one validates their reagents. What I wonder is whether the paper you cite has validated the specificity of the siRNA

Andy Resnick

I won't have access to the full article until Tuesday (access thru work), but based on the abstract, I don't understand why you say the article concerns validation of reagents. I read the abstract as validation of a method to measure the spatial distribution of expressed proteins by siRNA (alternatively, validation of the siRNA method as per Monique).

Why do you say "validates their reagents like antibodies"?

Monique

I don't find the paper impressive and they appear to ignore the fact that knockdown might not be efficient or has off-target effects. The idea to use siRNA to verify protein localization is not new.

The statement that absolutely no one validates their reagents is ridiculous, it makes me wonder what field you are in to have such a view. The statement that in industry the validation is absolutely required is not valid either since I mentioned that commercial antibodies can also be a-specific. I would applaud it if these antibody selling companies would validate their products.

Ygggdrasil

To play Devil's advocate, there are other uses for antibodies than immunofluoresence and often the antibodies that work well for immunofluorescence do not work well for these other applications (such as Western blotting). So, these 20% of commercially available antibodies that don't seem to work well aren't necessarily worthless (although from experience, I've come across plenty of commercially available antibodies that don't seem to work well for any purpose).

Andy Resnick

Sure- most manufacturers even suggest 'optimal' applications (for primary antibodies: ELISA, WB, IP, IF, etc.), see http://igene.invitrogen.com/isearch/antibody.do

It's also true that *negative* controls should *always* be performed to check against non-specific binding (for example). Lastly, since experimental systems vary so widely all protocols: immunohistochemistry, siRNA, transfection, etc, must be carefully optimized: what works on one cell type may not work on another.

The OP did not understand the content of the linked article.