SDS-PAGE gel resolution

Michael L. Sullivan mlsulliv at facstaff.wisc.edu
Thu Aug 3 10:44:49 EST 2000


Also, if you are expressing your protein in E. coli, you might spend more
time on sample prep to get rid of some of the DNA and other crud.  Some of
the quick protocols in product literature (like just boiling cells in
SDS-PAGE buffer) work fine when your expressed protein is a big fat band,
but don't make a clean enough gel to see and be sure of something that
isn't expressing well.

Mike



>"Martin Thompson" <bugeater at cyllene.uwa.edu.au> wrote in message
>news:39893D54.F6B402F2 at cyllene.uwa.edu.au...
>>
>> I was wondering if anyone knows what kind of factors influence how
>> 'fuzzy' bands are on a SDS-PAGE gel.  Currently I am having problems
>> with this and would like to know how to increase the sharpness or
>> resolution of these bands.  I suspect that this is leading to problems
>> with me seeing exactly where my protein is (its expression is quite
>> low).
>> Thanks in advance,
>> Marty.
>> --
>
>Marty,
>
>more form emperical than truely logical workings, I found the best way was
>to
>
>1) use the lowest volume of loading possible (use 6x loading buffer-needs
>heating to use)
>2) run sample into the stacking get at maximum volts/amps your gel can take
>3) run at the slowest practical speed for the rest of the gel (keep gel as
>cool as possible but wiuth enough volts/amps to keep the protein running
>down rather than across or out)
>4) run for 2-3 mins at maxium speed at the end. (snaps the bands togeather,
>especially useful for large protiens)
>
>Of course I am assuming if you are making the gel yourself you are using
>water saturated butan2ol to "smooth" the air/gel interface, and making sure
>the combs are clean as so the stacking gel is perfect.  Wash the wells out
>well before loading-I use a 20G blunted needle for this, with a 5 ml syringe
>filled with running buffer.
>
>JAne
> More practical than theoertical.
>
>
>Absolut Queer                             Karmically Challenged
>absolut-queer at DOTNOTDASHbigfoot.com
>
>Science is like being God, only I have time and budget constraints.


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