Re: PH of IHC (What pH?)

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From:"J. A. Kiernan" <>
To:Histonet <>
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On Sun, 2 Apr 2000, anita dudley wrote:

> Thanks to everyone who responded to my question about PH of IHC buffer.  
> There is a meter out in the clinical lab but I am going to purchase a 
> smaller one,

While on the subject of pH, it's worth noting that the usual 7.2-7.6
may not be optimal in many cases, especially for monoclonals. The
paper cited below (with short & inadequate summary by me) seems to
have something important to say.

Boenisch,T (1999): 
Diluent buffer ions and pH: Their influence on the
performance of monoclonal antibodies in immunohistochemistry. 
Appl. Immunohistochem. Mol. Morphol. 7, 300-306. 

Monoclonal antibodies (12 tested) work at higher dilution in 0.05M TRIS
pH 6.0 or 8.6 (varies with antibody) than at physiological pH. (NaCl also
disadvantageous) The 2 polyclonals tested were insensitive to the

It's also worth adding that you can't have a TRIS buffer below
pH 7.2. The TRIS/HCl system has buffering capacity only in the
range 7 to 9.5. A solution at pH 6 might contain TRIS but it
wouldn't be a buffer, and tiny chemical changes (such as adding
water) could change the pH significantly.

 John A. Kiernan,
 Department of Anatomy & Cell Biology,
 The University of Western Ontario,
 LONDON,  Canada  N6A 5C1

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