Methods Digest, Vol 81, Issue 2

Nikola Loncar via methods%40net.bio.net (by hemicar085 from yahoo.com)
Tue Feb 7 12:26:04 EST 2012


Hi there, 


I disagree with DK since molarity of buffer can not be calculated as a sum of two component. Lets take MES acetate as an example. 1 M buffer means that there is one mole of MES in 1 L of buffer whose pH is adjusted with acetic acid. 

Concerning tris hepes buffer - is it possible that your colleague meant that you can use either tris or hepes? otherwise, you can dissolve hepes to be 50 mM and adjust pH with concentrate solution of tris (tris solution is by itself very basic - pH ~11), or other way around but i can not remember any explanations why anyone would use that buffer. 


best wishes, 
N.L.



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Today's Topics:

   1. Tris-HEPES (Yvonne Couch)
   2. Re: Tris-HEPES (DK)


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Message: 1
Date: Mon, 6 Feb 2012 15:31:15 +0000
From: Yvonne Couch <yvonne.couch from pharm.ox.ac.uk>
Subject: Tris-HEPES
To: "methods from magpie.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
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    <FC9D052C4A50C04E8D6C54D2F870A5819380041B8F from EXMBX01.ad.oak.ox.ac.uk>
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Hi all,
I am trying to replicate the experiments of an ex-colleagues whose notes require '50mM tris-HEPES buffer (ph7.5)'. I am not sure whether the 50mM refers to the tris or the HEPES and was wondering whether anyone had any experience with this? I am using it as an extraction buffer for fresh tissue (the other components of which are sucrose and EDTA) and need to maintain the integrity of the enzymes within the tissue, rather than the cells. If anyone has any suggestions for a replacement for this buffer or any more details on how to make a tris-HEPES buffer, I would be most grateful.
Regards
Yvonne



------------------------------

Message: 2
Date: Tue, 07 Feb 2012 02:18:25 GMT
From: dk from no.email.thankstospam.net (DK)
Subject: Re: Tris-HEPES
To: methods from net.bio.net
Message-ID: <QR%Xq.9509$rV2.1695 from newsfe11.iad>

In article <mailman.222.1328552955.3721.methods from net.bio.net>, Yvonne Couch <yvonne.couch from pharm.ox.ac.uk> wrote:
>Hi all,
>I am trying to replicate the experiments of an ex-colleagues whose notes
> require '50mM tris-HEPES buffer (ph7.5)'. I am not sure whether the 50mM
> refers to the tris or the HEPES and was wondering whether anyone had any
> experience with this? 

There is plenty of ambiguity there but I'd think that it means that
the buffer is made with 25 mM of Tris and 25 mM of HEPES. 
At least that's how it works in our lab: a "1.0 M MES-Acetate"
is 0.5 M of each component. 

> has any suggestions for a replacement for this buffer 

It's going to be a very good buffer at 7.5. Lots of things could 
work as replacement though. 

>or any more details on
> how to make a tris-HEPES buffer, I would be most grateful.

Mix HEPES (free acid form) and Tris (free base form) and 
adjust pH to 7.5 with either NaOH or HCl (I am not sure what 
pH is going to be when mixing equal concentrations of 
HEPES and Tris). 

DK



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