Mari,

I agree with Thomas. You are making some significant protocol error.
Although most folks do not set up their confocals for single molecule
detection, it is possible to do so. Even under "typical" operating
conditions and a fluor that does not bleach too fast, confocals can
detect tens of fluor copies. Please elaborate on your staining
protocol as Thomas suggests.


>You don't say how you fixed and permeabilized your cells.  If the MHC
>is inside, you have to do something to allow the abs to penetrate
>into the cell.  The fixation procedure could denature the antigen or
>otherwise block the binding site, the permeablization method may not
>expose the binding site, the permeabilization method may extract the
>target antigen.  I don't have any data but I would have guessed that
>immunocytochem is more sensitive than IP.
>
>>Hello,
>>
>>I have now  perplexed results; I saw good amount of protein when I did
>>immunoprecipitation, but I cannot detect the protein by confocal
>>micromicroscope.  I am wondering whether IP is more sensitive than
>>microscopic observation, in general.  Of course, my staining system matters
>>here.  I used biotinylated Ab as an primary, then used
>>streptoavidin-conjugated either Cy3, Red-X or FITC.  I assume that I may
>>need brighter staining, so that I am considering to enhance the signal
>>(using TSA most probably - Thank you very much for your input!  It was me
>>who was asking about triple staining a several days ago).  Then hopefully I
>>can detect the protein also microscopically.
>>
>>Any information will be appreciated.  And thank you for your help/replies on
>>my last question.
>>
>>
>>Mari
>>
>>P.S. To be more specifically speaking, I am using anti-MHC class antibody
>>(28-14-8).  It is known that RMA-S cells produce class I molecules and they
>>can be detected by IP using this particular Ab, but the molecules does not
>>come out to cell surface under normal condition.  I use this cells as a
>>control of experiment and stained it.  There was no cell surface staining,
>>of course, but there was no tracellular staining as well.  And my testing
>>cells behaved the same.  So that's why I am wondering whether IP is
>>super-sensitive and confocal detection cannot be that sensitive  (otherwise,
>>it does not makes sense...)
>
>--
>Thomas E. Phillips, Ph.D.
>Associate Professor of Biological Sciences
>Director, Molecular Cytology Core Facility
>
>3 Tucker Hall
>Division of Biological Sciences
>University of Missouri
>Columbia, MO 65211-7400
>(573)-882-4712 (voice)
>(573)-882-0123 (fax)

--

Mario M. Moronne, Ph.D.
NanoMed Technologies
ph (510) 528-2400
FAX (510) 528-8076
Berkeley, CA
94706