Antibody concentrations....

Mario Roederer Roederer at Darwin.Stanford.EDU
Thu Jun 13 17:16:07 EST 1996


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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