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BMB anti-HA and immunofluorescence

Mike Rout rout at rockvax.rockefeller.edu
Wed Feb 15 15:53:03 EST 1995


In article <3hr910$5ko at charm.magnus.acs.ohio-state.edu>,
tchang at magnus.acs.ohio-state.edu (Tien-Hsien Chang) wrote:

>      After attempting a few channels to obtain anti-HA antibodies sold by
> BabCo, we are still unable to get any.  Thus, we were forced to use mouse
> anti-HA monoclonal from BMB.  This (expensive) antibody, although useful in
> Western analysis on our hands, appeared to have significantly high background
> in indirect immunofluorescence studies.  In several tries, even using
> preadsorbed (by yeast powder) BMB anti-HA, we still saw pretty obvious
> background staining in the negative control, i.e. strains which do not contain
> any HA-tagged proteins.  Our control experiments using different antibodies
> lighted up where they supposed to be.  So, the problem seems to be
derived from
> BMB's anti-HA per se.
> 
>      Is there anyone out there successfully using BMB's stuff for indirect
> immunofluorescence studies on S. cerevisiae?  If so, can you kindly offer
> suggestion to us?  Also, can someone who has BabCo's anti-HA kindly
offer a bit
> of that antibody?  Thanks in advance.
> 
>                               Tien-Hsien Chang
>                               Department of Molecular Genetics
>                               The Ohio State University
>                               484 West 12th Ave.
>                               Columbus, OH 43210
> 
>                               (614)-292-0631
>                               (614)-292-4466 [fax]
>                               chang.108 at osu.edu

We have used the anti-HA mAb on numerous occasions to visualize nuclear
pore complex proteins.  We found exactly the same problem (we have a batch
of the mAb from BAbCo);  we see a "speckly" background (mainly
cytoplasmic) staining.  We reduced this background (and often dramatically
increase the signal) by using much shorter fixation times in formaldehyde
(harvest the cells out of their medium by filtration, wash, and fix 3 - 5
min in 4%F, K-Phos/Mg, then proceed with cell wall digest - follow up
after this with a MeOH/Acetone post fix on slides.  Use 2% milk powder,
PBS, 0.1% Tween-20 for all antibody incubations and washes except the last
few washes prior to mounting the slides, when you wash in 1% BSA/PBS).  By
further similar fiddling around, we even got immunoEM to work with the HA
mAb (after a fashion!)  More details are in Wente et al., 1992, J. Cell
Biol. 119, 705-723.  The problem is, this reduction in background is at
the expense of morphological preservation and retention of soluble
cytoplasmic proteins.  To be honest, we've more or less given up on the HA
tag in favour of the protein A - IgG binding sites tag.  

Hope this is of some help - good luck, and feel free to email if there's
any problems.

Mike Rout

-- 
Mike Rout                            212 327-8098 (lab)
Laboratory of Cell Biology           212 327-8660 (fax)
The Rockefeller University/HHMI
1230 York Ave.
New York, NY 10021



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