SDS-problems

Bassie baslee at kabelfoon.nl
Thu Oct 21 11:28:10 EST 1999


Pedro Silva wrote:

> I have partially purified a membrane protein from a hyperthermophilic
> organism. The protein has been solubilized with 0.4% DOC, then
> precipitated w/ 15% ammonium sulfate and resuspended in 0.4% DOC
> -containing buffer.
> Then I tried to run a SDS-Phast Gel with it, but the protein did not
> migrate at all. I had boiled it for 10 min w/ SDS and
> beta-mercaptoethanol. It also did not work when I autoclaved the samples
> in the presence of 1 M urea or 1 M guanidinium prior to application onto
> the gel. Does anyone have any idea how to denature this stubborn
> protein?
>
> thanks!
> Pedro Silva

There are some things I hink of when reading your problem..
1: hyperthermophilic: That could mean that there are a great number of S-S
bonds in your protein and then the standard amount of beta-mercaptoethanol
(bME) might be insufficient..
Try to incubate your protein wit 10x the amount of bME at 70-80 C before the
standard SDS solution
2: the presence of DOC: The effects of SDS might be reduced due to the
presence of DOC as it stabilizes your protein significantly.
Try to increase the amount of SDS used in the incubation..
3: the presence of Ammonium Sulfate: Basically the same reason as for the
DOC..
Try to dialyse or PD10 your sample to remove the Amm.Sulf.

Succes..

---=== Bassie ===---
Biochem. since 1980




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