On 29 May 1995, Elke Greif wrote:
>> Hi! I am in the process of trying to perfect the method of 2D
> electrophoresis. My problems are many little ones which I haven't been able
> to quite figure out.
> 1) When the IEF is poured, the polymerization is OK. Once the tubes(2.5mm)
> have run overnight at 400v, the next morning I notice a tiny air bubble in
> almost all the tubes which was not there before. I degas my IEF solution.
If you are referring to an air pocket at the bottom end, that will not be
a big deal for a 2.5mm tube. However, if you find that air
bubbles/pockets form along the length of the tube it may be due to poor
cleaning of the tubes. Before you pour the gels make sure the tubes are
absolutely clean and dust-free. Soak the tubes in 3N H2SO4 overnight,
then get rid of the acid by sveral rinses with ddH2O, with intermittent
rinses using 95% EtOH. Once you are confident that the acid has been
washed off, dry the tubes in an oven, silanize them following the proper
procedure and dry them again. Store the tubes, if not used immediately,
in sealed bags.
> 2) When I pour the running gel of the second dimension, I overlay with
> water, let polymerize, then there is a small bump right in the middle on
> top of the gel.
Overlay with water-saturated n-butanol. Also reduce the vol. of 10% APS
added to: 1.33uL/mL gel solution. This will slow down polymerization
and allow the overlay to spread out evenly before the gel starts
polymerizing.
> 3) Pouring the upper gel of the second dimension, I insert a tooth for
> running the standard alongside the IEF and then once it has run, the
> standard in the middle has run at an angle so I don't really have the exact
> location of that particular standard.These standards are pre-stained high
> molecular weight from BRL. They didn't have an answer .
I normally do not use any comb for the 2nd dimension. What do you mean
by 'standard in the middle'? I load my standards as an 'agarose slice'
where a half-centimeter piece of 0.75% agarose contains the right amount
of the markers (already denatured etc.). The IEF gel and the marker slice
are placed on the 2nd dimension gel and sealed onto place by 0.5%agarose.
--- ----------------------------
Also, try to run a 1mm SDS gel, instead of thicker gels to acommodate the
2.5mm tube gel. If you have one of the plates beveled, it is not a
problem at all. Takes a little steadiness and one or two gels to get the
hang of it.
Any help to these problems would be greatly appreciated. >
>egreif at ccu.umanitoba.ca>> Thanks in advance.
>>
>>>>>>>>>>>>>>>>>>>>>>>>>>>
Hiranya S. Roychowdhury
Plant Genetic Engineering Lab.
Box 3GL, NM State Univ.
Las Cruces, NM 88003
Phone: (505) 646-5785
hroychow at nmsu.edu
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