Flow cytometry

Mark Kaplan mkaplan at mbcrr.harvard.edu
Wed Jul 26 10:16:31 EST 1995


In article <13488CA4F5F at prl.pulmonary.ubc.ca>,
DANDERSON at PRL.PULMONARY.UBC.CA wrote:

> The mouse MAb is excellent for staining virus, however, in murine 
> tissue the secondary gives the background.  This occurs both with 
> the conventional direct and indirect staining with respect to a labelled 
> 2' anti-mouse antibody.  

   The solution here would be to try another type of secondary Ab.  There
is tremendous variation in how companies prep their Ab reagents and some
may be cleaner than others.  Alternatively, if you have considerable
amounts of mAb, you can FITCylate or biotinylate the Ab directly.  The
former requires no 2°, the latter would use a streptavidin-fluorochrome
conjugate.


>The non-specificity of the rabbit antibody appears to be primarily in the
>rabbit antibody itself as there is no 2' Ab background staining.

   This can be a common problem in using rabbit antisera.  The best way to
overcome this is to purify the Ag-specific Abs.  this can be done by
running the sera over an Ag column and eluting only the binding Abs.  If
Ag quantity or purity is limiting, you can run a PAGE gel, transfer the
protein to a membrane and use specific cut out bands to bind your Abs. 
The first suggestion is far better in achieving both purity and
significant recovery.


Hope this is of some help.


Mark Kaplan
Dept. of Cancer Biology
Harvard School of Public Health



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