[Histonet] Re: Somewhat rhetorical question regarding antibody incubation time

From:"Johnson, Teri"

Dear Jackie,

I think it depends on the antibody. I found this explanation on antibody
equilibrium at the Chemicon website
(http://www.chemicon.com/resource/ANT101/a1.asp):

"The time taken to reach equilibrium is dependent on the rate of
diffusion and the affinity of the antibody for the antigen, and can vary
widely. The affinity constant for antibody-antigen binding can span a
wide range, extending from below 105 mol-1 to above 1012 mol-1. Affinity
constants can be affected by temperature, pH and solvent. Affinity
constants can be determined for monoclonal antibodies, but not for
polyclonal antibodies, as multiple bondings take place between
polyclonal antibodies and their antigens."

Some antibodies may react at room temp for one hour at 2x the standard
dilution (which is used at 4 degrees C). Other times you can use the
exact same concentration as you would overnight in the fridge, and your
staining is as intense, but with possibly more background.  If you're
trying to automate the antibody by bringing it to room temp, try a
variety of antibody concentrations and see if you can duplicate the
results you get from the refrigerated incubation.


Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, MO 64133

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