15% native gel shift expt.
(by lautys from gmail.com)
Wed May 20 21:42:00 EST 2009
hi Yan Sun,
I'm not an expert in this but putting me in your condition, I would to try
out using my friend's protocol, including using the sodium phosphoate
buffer. As a researcher, u know sometime things just work.
Another alternative is consult Bio-rad's (not sales representative)
technical group, they might able to troubleshoot your problem. If they
product worked once for you, there is no reason why it does work anymore.
wish you good luck.
On Thu, May 21, 2009 at 9:22 AM, yan sun <yansun2005 from gmail.com> wrote:
> Hi everybody, good evening!!
> I was bothered by this 15% native gel shift experiment for a while.
> I study the interaction between NC protein and HIV TAR DNA.
> The gel was ordered from Bio-rad. It worked very well once. I ordered more,
> but this time there is no Complex band show up (interaction complex).
> I did not change anything, look like the gel has problem, just like
> denatured gel. But my friends used my gel, of course study his NC with HIV1
> stem loop3 (different from my Tar DNA),his buffer is different from mine.
> The complex band shows up. Which means the gel itself did it's job.
> I felt quite confused. I made new running buffer (1X TBE), fresh DNA, fresh
> NC, new loading buffer(50 mM hepes,pH 7.5). Change one by one reaction
> condition each time, but none of them got me good results.
> My friends used sodium phosphoate buffer, 150mM NaCl. Actually it will
> the nonspecific binding more than my buffer, right? How come he got
> complex, but my is none? Since we used different Nucleic acids, of course
> the Kd values are different, there is nothing to compare.
> I am very very upset, could anybody experienced in this field give me some
> Thank you so much!
> Y. F.S.
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