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Alternatives to Laemli?

Alternatives to Laemli? - Protocols and Methods Forum

Alternatives to Laemli? - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 02-16-2004, 10:17 PM
MICHAEL L SULLIVAN
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Default Alternatives to Laemli?





Is there a point to including a stacking gel here, since the pH difference wouldn't be that great between the stacking/resolving gels? I thought that was the point of a stacking gel-- having the pH difference at the interface of the resolving/stacking gel. Of course, I've been puzzled how one can have precast gels, since presumably the pH difference couldn't be maintained upon storage, so maybe I'm wrong on this.

Mike

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  #2  
Old 02-16-2004, 10:55 PM
EK
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Default Alternatives to Laemli?


"MICHAEL L SULLIVAN" <[Only registered users see links. ].edu> wrote in message
news:[Only registered users see links. ].edu.. .
wouldn't be that great between the stacking/resolving gels? I thought that
was the point of a stacking gel-- having the pH difference at the interface
of the resolving/stacking gel. Of course, I've been puzzled how one can
have precast gels, since presumably the pH difference couldn't be maintained
upon storage, so maybe I'm wrong on this.

The Laemmli stacking system works by utilizing a difference in mobility of
glycine and SDS/protein at different pHs. At pH 6.8 glycine and proteins
have relatively equal mobilities through low percentage PAAG. However, when
both reach separating gel at pH 8.8, glycine picks up creating a charge
"vacuum" zone immediately behind the transition line between the two gel
layers. This charge vacuum slows down migration of proteins as they enter
the separating gel, causing a stacking effect. I hope I remember that
correctly.

As for the original post, I don't think SDS-based buffer would work for
studying protein/RNA complexes at all, simply because SDS would denature the
protein. There are non-denaturing buffer systems, like Tris-borate-EDTA
(TBE) that work fine for that purpose.


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  #3  
Old 02-16-2004, 10:57 PM
EK
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Default Alternatives to Laemli?


when

---- I will correct myself: its "immediately below the transition line" (in
vertical systems)

the


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  #4  
Old 02-16-2004, 11:05 PM
Wolfgang Schechinger
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Default Alternatives to Laemli?

I remember that a german company named Carl Roth used to sell a phosphate
based laemmli claiming phosphate had a smaller temperature coefficient and
thus yielded a more stable pH during the boiling step and thus prevented
some chemical proteolysis (to reduce the number of 'artificial' bands).

If someone is interested, I'll dig out the receipe.

Wo


At 16:57 16.02.2004 -0600, EK wrote:
Wolfgang Schechinger


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  #5  
Old 02-17-2004, 04:47 PM
Michael L. Sullivan
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Default Alternatives to Laemli?

>

I think another approach that people use when studying RNA binding
proteins is to do UV crosslinking/label transfer type protocols.
With that approach, a radio labeled RNA target is incubated with a
protein extract, RNA is UV crosslinked to the protein, and then
excess RNA is degraded enzymatically. This leaves proteins with
(hopefully) small pieces of labeled RNA attached which can then be
RUN on a regular SDS-PAGE gel and detected by autoradiography..

Mike
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Research Plant Molecular
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U.S. Dairy Forage Research Center
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Madison WI, 53706

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