Native gel and salt - (Jan/11/2008 )

Hi everybody,
I'm a new to this forum.
I read the topics dicussed in the forum and it seems very interesting and useful for me.

Anyways I would appreciate if anyone could give me some suggestions regarding protein precipitation during a native gel migration.
I work on a protein, that needs salts (~50mM NaCl minimum), to be stable. During the migration of my protein in a native gel, it appeared that it precipitates a few minutes after the start of the experiment. I think that Na Cl ions migrate faster than my protein, that's why I assume my protein precipitates during the migration...


Could anyone give me some tips or a protocole for native gel with salts. ALL kinds of suggestions are welcome.

Thanks
JB

Hi,

I am sorry to say that I dont have any answer to your question JB since I also have problems running native gels. I have a problem that after running the gel and staining it I realize that the bands are not sharp. This has never happened when I run SDS-PAGE gels. I use 4% stacing gel (pH 6.8, 0.125 M Tris-HCl) and 7.5% separating gel (pH 8.8, 0.375 M Tris-HCl). Running buffer is 0.25 M Tris-HCl, pH 8.0. Does anyone know if there is somenthing wrong in the gel or running buffer? This is the first time I am running native gels. Thanks.

Sarita

artero:

are you sure that your protein didn't denature in the gel due to heat? salt in the sample causes heating in the lane.

you can try to add the minimal amount of salt to the gel and electrode buffer. if you do then you will need to either cool the gel during the run and/or run slower to minimize the heating.

i haven't tried adding salt but have used other additives this way.


sarita:

your electrode buffer looks too concentrated to me. you can try the buffer from this post: ornstein and davis gel