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Electrophoretic Mobility Shift Assay Forum EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


what are they in my EMSA?

what are they in my EMSA? - Electrophoretic Mobility Shift Assay Forum

what are they in my EMSA? - EMSA Forum. Discuss the Electrophoretic Mobility Shift Assay protocols or technique and the assessment of DNA and RNA binding proteins here. Also post questions and troubleshooting about the shift assay.


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  #1  
Old 10-31-2008, 03:30 PM
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Default what are they in my EMSA?



Hi,can anybody help me find them out?
I'm investigating NF-KB in the kidney with EMSA using biotin-labeled probe.(Lightshift chemiluminescent EMSA kit,PIERCE).
The nuclear extraction from rat kidney are subjected to EMSA.it seems that nf-kb were found out in the image,but there exsist long bands at the top of the gel(???) which were unable to be competed with 200-fold unlabeled probe.what are they?
thax!
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Old 11-03-2008, 06:21 AM
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Default Re: what are they in my EMSA?

Do you mean the very faint bands on top of the major band? Are they just non-specific bindings to your biotin-labeled probes? Because nuclear extract might contain some other nuclear proteins with very high IP, and those proteins might bind to DNA just by electrostatics. Did you try to use higher salt conditions to minimize this interference?
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Old 11-04-2008, 03:43 AM
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Default Re: what are they in my EMSA?



Hello Andy, I agree if they could not be competed by specific probe they are non-specific aggregates of biotin-labeled protein. There are many large and small DNA binding proteins that bind to most DNAs.

Did you try to use non specific competitors in your binding reactions?

cheers
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Old 11-05-2008, 12:57 PM
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Default Re: what are they in my EMSA?

Thanks very much!
Hi blackangel and admin,the faint bands in two lanes appeared several times in my EMSA and hardly were eliminated.Because that the concentration of nuclear extractions from the rat kidney were about 2ug/UL,i didn't use higher salt conditions according to the instruction of PIERCE kit.The non specific competitors poly(di:dc)were added to the binding reactions(20ul) with 1ul .Is it possible that the faint bands were free porbes?
By the way,how to disperse the nuclear pellet after centrifuging(16000xg)?


,the concentration of the nuclear extractions was about 2ug/ul,so i didn't use higher salt conditions according to the instruction of PIERCE kit.
the non-specific competitors poly(di:dc) were added to the binding reactions with each
1ul

Is it possible that the faint bands in the two lanes were free probes?


thanx again!
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Old 11-06-2008, 01:49 AM
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Default Re: what are they in my EMSA?

what's the matter with my post?["Thank you for posting! Your post will not be visible until a moderator has approved it for posting."]
i can't give my post!
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Old 11-06-2008, 01:51 AM
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Default Re: what are they in my EMSA?

Hi blackangel and admin,the concentration of the nuclear extractions was about 2ug/ul,so i didn't use higher salt conditions according to the instruction of PIERCE kit.
the non-specific competitors poly(di:dc) were added to the binding reactions with each
1ul.Is it possible that the faint bands in the two lanes were free probes?By the way,how to disperse the nuclear pellet after centrifuging(1,6000xg)?
Thanx again!
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Old 11-06-2008, 01:55 AM
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Default Re: what are they in my EMSA?

hi blackangel and admin,the concentration of the nuclear extractions was about 2ug/ul,so i didn't use higher salt conditions according to the instruction of PIERCE kit.
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Old 11-06-2008, 01:56 AM
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Default Re: what are they in my EMSA?

the non-specific competitors poly(di:dc) were added to the binding reactions with each
1ul.Is it possible that the faint bands in the two lanes were free probes?By the way,how to disperse the nuclear pellet after centrifuging(1,6000xg)?
Thanx again!
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Old 11-06-2008, 01:57 AM
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Default Re: what are they in my EMSA?

The non-specific competitors poly(di:dc) were added to the binding reactions with each
1ul.
Is it possible that the faint bands in the two lanes were free probes?By the way,how to disperse the nuclear pellet after centrifuging(1,6000xg)?
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Old 11-06-2008, 08:21 AM
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Default Re: what are they in my EMSA?

Hello Andy,
sorry sometimes it thinks your posts are spam (if you have more than 10 posts) and they go into a waiting list until i approve them.

usually free probe is very small and runs to the bottom even on high percentage gels. How big is your probe in kb or bases?

Non-specific competitor is good to add. However how much protein are you adding from your nuclear extract?

After centrifuging you should re-suspend in suitable buffer for EMSA similar to your binding conditions. Have you optimized the binding conditions for your specific protein? What pH does it bind the most? What [salt]? What buffer did you use Hepes?

Sorry for the many questions however EMSA is quite specific to the protein and probe!

cheers
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