2-D gel trouble
logand at msdos.montpellier.inra.fr
Fri Jun 3 12:11:48 EST 1994
1. Is the current predicted by Bio-rad for the same number and size of tube gels
as you are using? Do you have the same concentrations of ampholytes? I notice in
the instructions for their system that I have looked out here, that they say to
use 100mM NaOH for the lower buffer - seems strange your 20mM is more normal but
perhaps their measured current is with that buffer thus the resistance would be
less between the electrodes and the current for a given fixed voltage would be
higher. Also why do you want to run at 200V this is not the specified voltage in
the instructions I have - 400V then a sharpening stage at 800V!
2. In which direction are they broad in the second dimension - vertical i.e.
resulting from the SDS-PAGE or horizontal and therefore more likely from the
IEF. I am also not too sure why you are running Mol. wgt markers on IEF tubes -
do you mean pI markers? Could be due to the current but insufficient info here.
3. Ah, with the other problems this could be the end result. I suggest you check
the IEF conditions by staining the IEF gel with Coomassie - this is very simple
and quick but you need to get the method from Gel Electrophoresis - A practical
Approach (that series - IRL Press I think) since I have forgotten the details
but its Coomassie then TCA. This will pin point the problem to one or other
dimension and after that you can investigate.
Hope this is of some use.
Sorry can't help more.
David C. Logan
Logand at montpellier.inra.fr
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