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Native Gel Question

Native Gel Question - Protocols and Methods Forum

Native Gel Question - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 10-11-2007, 09:05 PM
Arne Christensen
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Default Native Gel Question



I would like to run three different purified proteins on the same
native gel. They are all have a predicted isoelectric point below
7.0, thus a native gel run in basic conditions should suffice;
however, I am having trouble getting one protein with an isoelectric
point of 6.7 to enter the gel.

I am using an pH8.8 continuous gel (no stacking gel). I abandoned
using the pH 6.8 stacking gel because I thought it was too close to
the IP of my IP6.7 protein, and was the problem. I"ve also tried
adding BME to my IP6.7 protein to try and abolish some of the
tertiary structure which may influence the IP of the protein.

Is it possible to run a native gel under basic conditions, but jack
up the pH of the running gel and buffer system, to say pH9.5 to try
and make my predicted IP6.7 protein more negatively charged?

One complication is that I would ultimately like to use this system
to show that two of these proteins bind each other by running them as
a mixture on a native gel. So, raising the pH too much may interfere
with this assay. Are there any other ways to make a protein more
negative without interfering with its capacity to interact with
another protein?

Thanks.
AC
--

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  #2  
Old 10-12-2007, 12:58 AM
DK
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Default Native Gel Question

Arne Christensen <[Only registered users see links. ]> wrote:

Something I always wanted to try but never got around to:
use 0.1 percent sodium deoxycholate in place of SDS in the
standard Laemmly recipe. This is certainly not denaturing but
should get just about any protein move briskly.

DK

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  #3  
Old 10-12-2007, 09:02 AM
corona
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Default Native Gel Question



Such an experiment would not be conclusive. You need to do some sort of pull
down assay or flourescence detection (e.g., split GFP).



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  #4  
Old 10-22-2007, 01:30 PM
Dr Engelbert Buxbaum
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Default Native Gel Question

Am 11.10.2007, 17:05 Uhr, schrieb Arne Christensen <[Only registered users see links. ]>:



Shure, you can freely select the pH at which you run your gel, this is
possible even with discontinuous gels. You need an appropriate buffer
system, which you can select on [Only registered users see links. ].

The relevant literature is:

@article{Jov-73a,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic Zone Electrophoresis. {I}. {S}teady-State
Moving-Boundary Systems Formed by Different Electrolyte Combinations},
JOURNAL= {Biochemistry},
VOLUME= {12},
YEAR= {1973},
PAGES= {871-879},
LANGUAGE= {engl}
}

@article{Jov-73b,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic Zone Electrophoresis. {II}. Design of
Integrated Discontinuous Buffer Systems for Analytical and Preparative
Fractionation},
JOURNAL= {Biochemistry},
VOLUME= {12},
YEAR= {1973},
PAGES= {879-890},
LANGUAGE= {engl}
}

@article{Jov-73c,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic Zone Electrophoresis. {III}. {F}urther
Analysis and New Forms of Discontinuous Buffer Systems},
JOURNAL= {Biochemistry},
VOLUME= {12},
YEAR= {1973},
PAGES= {890-898},
LANGUAGE= {engl}
}

@article{Jov-73d,
AUTHOR= {T.M. Jovin},
TITLE= {Multiphasic zone electrophoresis. {IV} {D}esign and
analysis of discontinuous buffer systems with a digital computer},
JOURNAL= {Ann. N.Y. Acad. Sci.},
VOLUME= {209},
YEAR= {1973},
PAGES= {477-496},
LANGUAGE= {engl}
}
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