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EMSA - Band doesn't shift with poly dI-dC


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#1 N_L

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Posted 31 August 2009 - 06:34 PM

Hi all,
I have been doing EMSA using Pierce kit for 4 months. My band shifted without adding poly dI-dC.
But with poly dI-dC, I didn't see no shift at all. I add 50ng/ul final concentration for poly dI-dC. I used 4ug of nuclear extract.
Can I leave this non-specific competitor in my reaction and go on to supershift?

I labeled my oligo with Pierce 3' end biotin labeling kit. I got the nice free probe every time.

My protocol is
- Incubate reaction at RT for 20 min (buffer, MgCl, glycerol, poly dI-dC in each reaction)
- 6% non-denaturing gel
- Run and transfer to Millipore nylon membrane with 0.5%TBE, (pre-chilled)
- detect biotin labeled oligo with non-radioactive Pierce kits.

Thanks in advance for replying.

Edited by N_L, 31 August 2009 - 06:35 PM.


#2 Dr Teeth

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Posted 01 September 2009 - 05:11 AM

Hi all,
I have been doing EMSA using Pierce kit for 4 months. My band shifted without adding poly dI-dC.
But with poly dI-dC, I didn't see no shift at all. I add 50ng/ul final concentration for poly dI-dC. I used 4ug of nuclear extract.
Can I leave this non-specific competitor in my reaction and go on to supershift?

I labeled my oligo with Pierce 3' end biotin labeling kit. I got the nice free probe every time.

My protocol is
- Incubate reaction at RT for 20 min (buffer, MgCl, glycerol, poly dI-dC in each reaction)
- 6% non-denaturing gel
- Run and transfer to Millipore nylon membrane with 0.5%TBE, (pre-chilled)
- detect biotin labeled oligo with non-radioactive Pierce kits.

Thanks in advance for replying.


If you are seeing a band without poly dI-dC, but no band with poly dI-dC, then the band you are seeing is not "your band," but a non-specific shift, thus, the point of using poly dI-dC---to remove non-specific bands. Something in your extract can associate with poly dI-dC, probably not your intended factor. Trying to do supershifts would probably be a waste of time and resources. Did your kit come with positive and negative controls. If so, how do they look? If not, you need to acquire some.

Science is simply common sense at its best that is rigidly accurate in observation and merciless to fallacy in logic.
Thomas Henry Huxley

#3 N_L

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Posted 02 September 2009 - 07:55 PM

If you are seeing a band without poly dI-dC, but no band with poly dI-dC, then the band you are seeing is not "your band," but a non-specific shift, thus, the point of using poly dI-dC---to remove non-specific bands. Something in your extract can associate with poly dI-dC, probably not your intended factor. Trying to do supershifts would probably be a waste of time and resources. Did your kit come with positive and negative controls. If so, how do they look? If not, you need to acquire some.


I got shift band with positive control coming along with Pierce kit. It was just that my designed oligo didn't moved up at all.
Maybe I still need to optimize the condition or my oligo is not the right one.

Thanks for the advice, anyway.

cheers




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