Immunochemistry question (probably dumb)

Jayakumar, R via (by R.Jayakumar from
Tue Jun 21 15:21:54 EST 2011

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From: methods-bounces from [methods-bounces from] On Behalf Of Peter Ellis [pjie2 from]
Sent: Tuesday, June 21, 2011 6:53 AM
To: methods from
Subject: Re: Immunochemistry question (probably dumb)

On Mon, 20 Jun 2011, DK wrote:
> But no matter how messy all of this is and what kind of complexes did
> eventually from at their particular dilutions, any claim of being able to
> distinguish proteins A and B is bound to be total unadulterated bullshit.
> Because no matter what, there ALWAYS be some complexes that will
> recognize both A and B. Must be. Under the best scenario possible,
> the complexes will be able to recognize only one of the proteins, failing
> to recognize the second one due to the lack of sensitivity (too low
> concentration). If not that, it will be BOTH proteins stained with both
> Cy5 and FITC. And no one could tell which one it is that is really
> detected.

That's what I thought. Their figure shows a very clear disjoint
distribution, with 50% of sperm positive for A and the other 50% positive
for B, with no apparent colocalisation (or antibody cross-talk). I just
don't see how that's remotely possible with the materials as given.

I can't verify the results myself since their primary antibody is
in-house.  In any case, the context is that I'm writing a review, and I
don't think I can reasonably be expected to go and replicate the results
for all the papers I'm citing!

The relevant bit of the materials and methods covering secondary AB
staining is as follows:

> Slides were washed three times in PBS-T, followed by 1 h incubation in
> fluorescein-conjugated goat anti-rabbit IgG diluted 1 : 200 in tissue
> sections or Cy5-conjugated goat anti-rabbit and fluorescein-conjugated
> rabbit anti-goat IgG (diluted 1 : 200) in sperm at room temperature.

The "in tissue sections" and "in sperm" part should I think say *for*
tissue sections / sperm respectively - they showed tissue sections stained
for A and B individually, and then sperm smears co-stained for both A and
B simultaneously.

I've contacted the authors to see if it's a simple error - i.e. they
actually used some other species for one of the secondaries.  We shall see
what transpires.

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