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______________________________ Reply Separator _________________________________
Subject: Antibody concentrations....
Author: Roederer@Beadle.Stanford.EDU at INTERNET
Date: 14/06/96 03:27
In regards to a recent question about a source of antibodies, Dr Hill replied:
> For flow, we use anti MET antibodies From UBI, catalog
> 05-237 (clone DO-24), the one listed for IPs. It works
> at 1 - 1.5 ug /10E6 cells
> (need to use a secondary for the fluorescent label).
I just want to add my annual reminder that the concentration of antibody to use
in a staining mixture is essentially independent of the number of cells being
stained, but is completely dependent on the stain volume! 1 ug of antibody is
more than enough for (in generaly) well over 10^8 cells expressing a typical
antigen--thus, it doesn't matter if you have 10^5, 10^6, or 10^7 cells in your
tube! Indeed, many antibodies will stain equally well at 10^8 cells (in, for
instance, 100 ul volume).
On the other hand, a reagent titred to be used in a 50ul stain volume may not
stain very well in 500 ul.
Specifically: 1 ug of antibody (IgG) is about 3.6 x 10^12 molecules--which (in
divalent fashion) binds 7.2x10^12 molecules of antigen. A typical antigen may
be present in concentrations of 10^4 to 10^5 per cell, meaning that 1 ug of
antibody would be enough to stain 7.2 x 10^7 to 7.2 x 10^8 cells. And, if only
10% of the cells express an antigen, you can multiply this by another factor of
10!
mr
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