Protocol Online logo
Top : New Forum Archives (2009-): : Molecular Biology

EMSA - Band doesn't shift with poly dI-dC - (Aug/31/2009 )

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.

-N_L-

N_L on Aug 31 2009, 10:34 PM said:

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.

-Dr Teeth-

Dr Teeth on Sep 1 2009, 09:11 PM said:

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

-N_L-