2 replies to this topic

[N_L]

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.

[Dr Teeth]

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.

[N_L]

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