Molecular cloning -Sambrook et al/ Tris base vs TRIZMA

Hiranya Roychowdhury hroychow at NMSU.EDU
Tue Jun 6 15:05:35 EST 1995


On Tue, 6 Jun 1995, Pamela Norton wrote:

> In article <Pine.A32.3.91.950605144847.56848B-100000 at paris>,
> hroychow at NMSU.EDU (Hiranya Roychowdhury) wrote:
> 
> > On 5 Jun 1995, T.S. Pillay wrote:
> > 
> > > In the electrophoresis section in Molecular Cloning by Sambrook et al 
> > > (a.k.a. the "Maniatis")  it says not to use TRIZMA for SDS PAGE
> > > electrophoresis buffers but Tris Base which should be pH'd to the
> > > appropriate pH instead of using a mixture of Trisbase and Tris-HCl.  Does
> > > anyone know the reason for this.
> > > 
> > But I thought Tris base and Trizma were the same. The compound used for
> > electrophoresis is tris(hydroxymethyl)aminomethane which, from Sigma,
> > comes as TRIZMA (base). The TrisHCl is simply the hydrocloride of the
> > above and is extremely acidic. 
> ...
> > 	I have never come across any protocol where the Tris buffer is
> > prepared by mixing Tris base and hydrochloride to obtain a specific pH. I 
> > am not sure what the authors in Sambrook et al. meant, but I will not be 
> > surprised if it is a mistake. There are a few very obvious yet serious 
> > mistakes in that manual. The question is that whether, by using Trizma 
> > base, anybody has encountered any problem in SDS-PAGE!
>         ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ 
> 				TIn response to the above:
> 
> 	         YES!!!!!! 
> 
>     I wasted over a month trying to figure out what the heck had gone wrong
> with my SDS-PAGE gels after setting up a new lab. Gels ran slowly, with odd
> voltage/current ratios, bands were fuzzy, resolution was poor in a critical
> mol. weight range (ca. 60 kDa). Problems were solved by formulating all the
> buffers with Tris base (we get it from Gibco/BRL, no affiliation,
> presumably other sources work as well) instead of Trizma. I too was
> skeptical but tried this as a last resort based on the comment in Sambrook
> et al. The difference was astounding. 
>     
>     However, I still don't know the correct answer to the original poster's
> question, but I believe the Cl- ion concentration is the critical thing. 
> 
>     Hope this helps prevent someone from wasting their time,
> 
> 													Pam Norton
> 
> -- 
> Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
> Thomas Jefferson University
> Philadelphia, PA 19107           p_norton at lac.jci.tju.edu
> 

I have used Trizma constantly for the last 12 years and never encountered 
any problem with it. However we once had some trouble (about 5 years 
back) with a bad batch of Glycine. Sigma sells another batch of 
Tris(hydroxymethyl)aminomethane under the name of Sigma 7-9 (or something 
like that), which is bad for electrophoresis. If there are more than the 
acceptable amount of other impurities in the Electrophoresis buffer, it 
will cause the run to have higher current and the run will be slow and 
there will be heating problem etc. etc. ultimately leading to poor 
resolution of the polypeptides. 


			>>>>>>>>>>>>>>>>>>>>>>>>>>>
			  Hiranya S. Roychowdhury
   			  Plant Genetic Engineering Lab.
			  Box 3GL, NM State Univ.
			  Las Cruces, NM 88003
			  Phone: (505) 646-5785
			  hroychow at nmsu.edu
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