15% native gel shift expt.

lautys via methods%40net.bio.net (by lautys from gmail.com)
Wed May 20 22:25:29 EST 2009


haha! yea, same thing with me, i always have first trial run very well and
struggle for the subsequent run...

anyway, if what u bought is not designed for gel shift assay, u might what
to switch to a proper product to ease things out.

cheerLau

On Thu, May 21, 2009 at 11:04 AM, yan sun <yansun2005 from gmail.com> wrote:

> 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.
>>> _______________________________________________
>>> Methods mailing list
>>> Methods from net.bio.net
>>> http://www.bio.net/biomail/listinfo/methods
>>>
>>
>>
>
>
> --
> Y. F.S.
>


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