dilution of antibody

Bruce Edwards BEdwards at salud.unm.edu
Tue Feb 29 17:33:31 EST 2000


You might be interested in a version of this experiment that we published back in 89:
Edwards, B.S., Shopp, G.M.  Efficient use of monoclonal antibodies for
immunofluorescence.  Cytometry 10:94-97, 1989.

 - Bruce

>>> "Donnenberg, Albert" <donnenbergad at MSX.UPMC.EDU> 02/28/00 09:37AM >>>

Joost,
I think that you should just do the experiment (we have).  Add graded
numbers of cells to microtiter wells (100K to 10M per well).  Stain your
cells as dry pellets (obtained by spinning the plates at 700 RPM
[~80xg]and
then flicking and blotting) using graded amounts of antibody (1 - 10
microliters).  You will be amazed.

Albert

-----Original Message-----
From: Joost Schuitemaker [mailto:J.H.Schuitemaker at amc.uva.nl]
Sent: Thursday, February 24, 2000 3:35 AM
To: cyto-inbox
Subject: RE: dilution of antibody



Dear Albert,

Sorry, but I don't agree with you. When you should look at a marker 80% of
your cells is positive for. And you determine the amount of antibody
(= your
concentration) for 300.000 cells. It is not possible to get even a
peak far
from your negative peak when you stain 5 million cells. This means
that you
throw away at least 5 times the amount of antibody when you stain 300.000
cells. Because you determined (titrated) your antibody for 5 million
cells.
And the definition of concentration is the amount of Ab in a certain
volume.
So when you got 100.000 cells and need 0.01 µg of Ab to stain them you can
add this in 50 µl or in 5 ml. The only difference is the timeperiod
it will
take to get your Ab on the cells. Or am I mistaken?

Best regards,

Joost Schuitemaker





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