Here is my updated protocol that does not require heating the protein sample in 6M GuHCl (3/2016): Take protein sample in 6M GuHCl and shear DNA with a needle/syringe, then take 10 uL and add to it 60 uL H2O, next add 40 uL 4X SDS/PAGE sample buffer (Tris-Glycine buffer system) and then add 30 uL of 1X SDS/PAGE running buffer (final volume 140 uL), mix gently and load sample to the bottom of the well using a gel loading tip. Sample will look very cloudy but loads fine with romm temperature running buffer (20-24oC) "as long as you do not pre-cool the running buffer in the gel rig", once samples are loaded then add an ice pack to the running buffer. Run samples until the dye is just in the gel, then turn off the power supply and flush out the wells to remove the GuHCl. Turn power back on and run as normal. Samples in 6M GuHCl, when loaded this way, will migrate with the same protein loaded without 6M GuHCl. I have only tested this on a Tris-Glycine buffer system.
I usually precipitate the samples (have to be diluted to < 2 M GuHCl first) and resuspend them in 8 M urea. Then add concentrated sample buffer, heat, etc.
Yes, you can run 6 GuHCl samples in SDS-PAGE as you describe, but it is cumbersome, has to be done quickly, and the sample is diluted 10-fold.
Arjan,
But protein samples in 6M GuHCl samples are compatible with SDS/PAGE if you prepare them properly (see above).
I usually precipitate the samples (have to be diluted to < 2 M GuHCl first) and resuspend them in 8 M urea. Then add concentrated sample buffer, heat, etc.
Yes, you can run 6 GuHCl samples in SDS-PAGE as you describe, but it is cumbersome, has to be done quickly, and the sample is diluted 10-fold.
Alejandro,
If you use gels with 50 uL wells you can load ~3-4 uL of the original sample, which for me is actually too much for some samples.
That's the problem, that you have to start adding constraints (work fast, use large wells, etc). That's why I feel that investing time in precipitating the samples first affords you flexibility and convenience. Your mileage, of course, may vary...
Mileage does vary, this is a quick and easy protocol for me, that is why I posted it!
Oh! I see, sorry, my bad; I did not realize you were contributing a tip and assumed it was some sort of question...
Hi Gary, I am trying to do this right now with a very high molecular weight glycoprotein. I have eluted this glycoprotein from the Protein G-bound antibody using 6 M guanidine hydrochloride. Normally when I run this glycoprotein there is a smear from 250 to 150 kDa on the gel. Can I use your method with this high molecular weight sample too? And are there any other little details to be careful of? My sample is really precious so I want to use one best method for it.
Alejandro, could you please share your protocol as well here?
Thank you so much
Dialyze your sample in a dialysis membrane against 8M urea. A 2-4 hr dialysis will do. Hope that helps.
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