SDS-PAGE SAMPLES

davesmith at bioch.tamu.edu davesmith at bioch.tamu.edu
Tue Dec 10 19:32:50 EST 1996


KAREN <MJFASKH at FS1.CE.UMIST.AC.UK> wrote:

>I'm trying to run protein samples which contain 6 M guanidine HCl on SDS
>PAGE gels, however when I add the sample buffer the sample precipitates
>and if I try to load it on the gel I don't get anything useful back! Can
>anyone suggest a way to get these samples on a gel short of actually
>dialysing them first?

>Alternatively, is there any way of dialysing lots of small samples
>fairly easily without the protein sticking to the membrane?

>Any ideas?

>Karen


Karen,
I have two suggestions.

You might try TCA precipitation:

First mix:
20 uL sample in 6M GdmHCl
400uL H2O
(the GdmHCl has to be diluted or it will precipitate)

Add 400 uL 10% TCA, vortex, incubate 20 min on ice and vortex again.
Spin 15 min in a microfuge.

Wash with 100 uL absolute EtOH (ice cold) and spin again.
Aspirate and air dry.
Resuspend the pellet in SDS loading buffer.

Unfortunately for us this method has an apparent molecular weight
cutoff of about 14 kDa--meaning we saw no bands on the gel below about
14 kDa.  Our protein runs at 8 kDa. :^0  Just couldn't get away from
the GdmHCl.

I was forced to dialyze my samples, so I cut up some 1.5 mL eppy tubes
for spite---not really.

I closed several eppy tubes and took a razor blade and cut off the
bottom of the tubes even/flush with the lower edge of the cap.

Open them back up and you have "microdialysis" containers/closures.
Put your sample (up to about 100 uL) in the cap.  Place a generous,
but small piece of dialysis membrane (your choice of MWCO) across the
top and carefully snap what's left of the "tube" over the cap.

Dialyze away with the "right side up".  Certainly not the most
efficient method of dialysis, but it works.

The tricky part was getting the sample out.  I discovered a use for
the box full of old tuberculin syringes and needles on the shelf.
Works like a charm.


Hope this helps!
Dave.




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