Aaron,
What do you mean by focusing for 7 hours at 750 volts? Are you using IPG
strips? Are you using Bio-Rad's IEF cell? If you are, then the standard
IEF protocol involves over 20000 volt-hours with the majority of the time
spent running 4000 volts through the 7 cm strips. You are describing using
5250 volt-hours and low overall voltage. To get maximum resolution in the
first dimension you have to have high voltage. Protein load should not
impinge on your resolving power of the Bio-Rad standards.
You should not need it for these standards, but for really, really good
resolution, micro-dialyze your samples against good quality water before
putting the samples into your IEF buffered solution. Micro-dialyzation is
the cat's pajamas for high resolution 2-D gels.
Jim Lawrence
Pioneer Hi-Bred, Intl.
Johnston, IA 50131
515-270-5909
-----Original Message-----
From: Aaron Downs [mailto:aaron_downs@thelabrat.com]
Sent: Monday, March 05, 2001 12:56 PM
To: Recipients of ABRF List
Subject: 2-D Gel Problem
hello all,
i'm working on a Bio-Rad mini 2-D gel system. we just got it in recently
and are trying to get our protocol down. we are using Bio-Rad's 2-D protein
markers as our guide and visualizing using silver stain (Bio-Rad too).
however, we seem to be having a problem resolving the more basic proteins of
this marker cocktail (>pH 6.0), while the more acidic proteins are fine. to
troubleshoot this, we have done:
1.) increased focusing time to 7 hours at 750 V.
2.) decreased protein load
do you kind people have any other suggestions for me to resolve these more
basic proteins better while keeping the same quality of acidic resolution?
i would really appreciate it.
Aaron Downs
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