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Western Blot Buffer Composition, why?

Western Blot Buffer Composition, why? - Protocols and Methods Forum

Western Blot Buffer Composition, why? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 01-28-2005, 09:01 AM
news.rrz.uni-hamburg.de
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Default Western Blot Buffer Composition, why?



Hi everybody,
One of my students asked me about the composition (especially methanol) of
the transfer buffers.
Has anyone a paper or a link where I can find the explanation?

Thank you very much
MA Bonetti


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  #2  
Old 01-29-2005, 12:46 AM
D.K.
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Default Western Blot Buffer Composition, why?

In article <ctcuu9$6iv$[Only registered users see links. ].uni-hamburg.de>, "news.rrz.uni-hamburg.de" <[Only registered users see links. ]> wrote:

Tris-Glycine: no good reason. I think the correct explanation is historical:
Towbin took 2X diluted SDS-PAGE running buffer without SDS and it
worked. As long as the stuff conducts but does not heat much (e.g.,
low moving ions), pH is high enough and the buffer is good enough,
anything should work. There are certainly other alternatives, like
(bi)carbonate or CAPS.

Methanol: To decrease polarity of the solution and to keep proteins
denatured. Both factors promote strong binding of ptoteins to the
hydrophobic membrane.

DK

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Old 01-29-2005, 02:28 AM
axexango@hotmail.com
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Default Western Blot Buffer Composition, why?


news.rrz.uni-hamburg.de wrote:
methanol) of

I know Amersham have a manual inclosed in the box with the
western membranes that give out the reference to that question. If I
remember correctly, PDVF membranes require methanol in their transfer
buffer but not nitrocellulose ones. Check in your suppliers box, see
if they have that manual.

HTH

Axexango

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  #4  
Old 02-01-2005, 08:55 AM
Dr Engelbert Buxbaum
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Default Western Blot Buffer Composition, why?

news.rrz.uni-hamburg.de wrote:


Dunn, Anal. Biochem. 157 (1986) 1144-153
Towbin et al., PNAS 76 (1979) 4350

and any decent textbook of protein chemistry. The topic has also been
extensively discussed in this usenet group and in bionet.molbio.proteins
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  #5  
Old 02-02-2005, 09:42 PM
Kyle Legate
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Default Western Blot Buffer Composition, why?

[Only registered users see links. ] wrote:

PVDF only requires that you wet it with methanol before soaking it in
transfer buffer, to make it hydrophilic. Once wet initially with
methanol, no further treatment with methanol is required.
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