15% native gel shift expt.

yan sun via methods%40net.bio.net (by yansun2005 from gmail.com)
Wed May 20 22:04:25 EST 2009


Hi Lau,

1) Thank you for your suggestion. I might try my friend's protocol.

2) I did consult with bio-rad's technical group. They said the native gel is
not designed for gel shift assay. They suggested me to use 0.5XTBE instead
of 1X TBE. They said high ionic strength might disturb the binding. I tried
0.5 X TBE, It did not work neither.

 Yeah, it was pretty confused. It is kind of ”luck" thing, all of
sudden, you could not  repeat what you did, even did well many times before.



Best,Yan




On Wed, May 20, 2009 at 10:42 PM, lautys <lautys from gmail.com> wrote:

> 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.
>
> regards,
> Lau
>
>   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
>> 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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>> Methods mailing list
>> Methods from net.bio.net
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>>
>
>


-- 
Y. F.S.


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