unknown concetration antibody Re: [Histonet] Negative control IHC

From:Gayle Callis


If the vendor can't provide a concentration (shame on them!), we try to use 
the IgG matching the host of the antibody. Ascites is another story.  This 
is always a guessing game, but PBS buffer is still never used as a negative 
control. Maybe with a polyclonal, normal serum could be used or an 
irrelevant antibody - a dilemma to be sure and a choice one has to make.

We had one antibody where we decided to use normal serum at same 
concentration as polyclonal even though that is not a choice we like very 
much.   The vendor (some obscure company) said the antibody was just 
antisera,  and nothing about purification.   Frustration ran high, our 
guess will be as good as the next person's.

Here is an email I kept from Deena Hepburn on how to determine protein 
concentration on ascites and antibodies, and may be important with a 
pending publication.

Here is the information for the protein assay I promised.    I saw this one 
and it looked like the easiest to do. There are no reagents to prepare.  I 
usually use the BCA or Comassie kit assay but they are a bit more 
complicated.  You will also need to purchase the standards.  For 
antibodies, I would buy the pre-diluted Protein Assay standard : Bovine 
Gamma Globulin Set.

With this assay you will need to dilute your ascites quite a bit.  Assuming 
your ascites is about 10mg/ml, I would do a dilution series
something like:
1:10, 1:100, 1:250, 1:500, 1:1000, 1:2500, 1:5000 (or something with a wide 
range - you should do a range to make sure you are on the curve).  You 
should read them on a plate reader but for a rough guess you can compare 
the  dilution with the standard curve to match colors.

For example:
If color of your 1:100 dilution matches the color of the 125ug/ml standard 
your concentration would be approximately 12.5mg/ml

125ug/ml (protein standard) X 100 (dilution factor) = 12,500ug/ml or 12.5mg/ml

Company: Pierce   Web address: www.piercenet.com

http://www.piercenet.com/products/browse.cfm?fldID=02020110  (assay)

http://www.piercenet.com/products/browse.cfm?fldID=02020108 (standards)

Feel free to call me if you need any additional help.

Deena Hepburn
Asst. Senior Biologist
Eli Lilly and Co.
Indianapolis, In 46285

Gayle Callis HTL, HT, MT(ASCP)
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717

Some At 12:54 PM 6/18/2007, you wrote:
>What do you do when you have a polyclonal antibody for which the vendor
>has not determined an Ig concentration?  It's easy to match
>concentration and isotype for a monoclonal but can be impossible for a
>polyclonal.  If we have an Ig concentration, we will match with Rabbit
>IgG.  If not, do you count on a buffer control, irrelevant antibody (not
>concentration matched), or normal sera?  Lots of choices, not sure what
>is the best for this situation.
>Tim Coskran

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