15% native gel shift expt.

yan sun via methods%40net.bio.net (by yansun2005 from gmail.com)
Wed May 20 20:22:37 EST 2009


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 break
the nonspecific binding more than my buffer, right? How come he got reaction
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
thoughts??
Thank you so much!

-- 
Y. F.S.


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