Coating-pH ?

Steve Hillerman Stephen_M_Hillerman at groton.pfizer.com
Thu Feb 22 10:20:10 EST 1996


Just adding my 2 cents.  Have you tried using 0.1M Na2CO3 at pH 9.6 as 
the buffer for your MAb's.  I have used it for polyclonals and have 
gotten very good binding.

Steve

-------------- next part --------------
From: haviland at KIDS.WUSTL.EDU ("David L. Haviland, Ph.D.")
Newsgroups: bionet.molbio.methds-reagnts
Subject: Re: Coating-pH ?
Date: 20 Feb 1996 11:06:20 -0800
Organization: BIOSCI International Newsgroups for Molecular Biology
Message-ID: <Pine.PMDF.3.91.960220124731.564216868A-100000 at kids.wustl.edu>
References: <Stephen_Lasky-0802960854100001 at cis-ts2-slip3.cis.brown.edu>

On Thu, 8 Feb 1996, Stephen R. Lasky wrote:

> In article <Pine.PMDF.3.91.960205122238.564180252B-100000 at kids.wustl.edu>,
> haviland at KIDS.WUSTL.EDU ("David L. Haviland, Ph.D.") wrote:
> > 
>  >After a few control assays, we used (and I continue to 
> > use) a 100mM Tris buffer pH9.5.  
> 
> David, just a question on this pH.   I thought the pKa for tris was a
> little above 8 so it wouldn't have much buffering capacity at pH 9.5.  Why
> not use a buffering agent that actually buffers at pH 9.5?  (I think that
> glycine has a pKa at 9.6 so it should actually buffer at that pH.)

Stephen: 

My regrets in taking so long to reply.  I've been in and out of town. To
your point: Yes, I agree that glycine would likely be a better buffer.
However, in discussing a "new" coating buffer we became concerned that if
something like glycine were used at an alkaline pH, would it not have the
potential to coat the plate itself and effectively block what we really
wanted to coat the plate with?  So without much experimentation, we never
considered glycine or much else.  Instead, we opted to "bend" Tris out of
its range, extrapolating off the Isco tables and set it up for pH 9.5 at
4'C.  I do periodically check it and it has held the pH for over six month
+/-0.1 pH unit and has worked fine for the last 12 years I've been doing
any sort of Ab detection/quantitation.  We've used the buffer to coat
MAbs, purified polyclonal Abs (goat and sheep), and peptides as small as
12-mers.  There probably is a better buffer, but if it works... do you
really want to fix it? Maybe I'll find an undergrad to test different
buffers ::grin::. 

However, for Alkalaine Phosphatase ELISAs, I DO use glycine pH 9.5 with 
Mg/Zn.  I use a sterilized form of this for alk-phosing vectors as I 
remember reading somewhere (in the fine print of a mol-bio manual, Perbal 
I believe) that Tris can inhibit Alk-Phosphatase.  Besides, glycine is 
easier to work with than ethanolamine (don't care for reagents that fume 
when one tries to pH them).  

David
p.s. - I agree with your sig, btw.
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