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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.


Gel shift of ternary complexes.

Gel shift of ternary complexes. - Electrophoretic Mobility Shift Assay Forum

Gel shift of ternary complexes. - 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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Old 07-24-2008, 02:40 PM
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Default Gel shift of ternary complexes.



Hey...
Does anyone here have any experience with setting up transcription reactions with crude extracts and trying to shift the ternary complexes on a native gel ?? I'd be interested in discussing the conditions under which u run ur gels.
Thanx.
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Old 07-24-2008, 06:24 PM
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Default Re: Gel shift of ternary complexes.

If you google gel shift protocols you should get a lot of educational reading.

Do you mean in vitro translation reactions with wheat germ or retic lysates?

Your best bet is to find some articles specific for the proteins you are working with other wise you could spend a good deal of time optimizing the binding conditions.

If you can give more details on the proteins and what you are trying to shift, I would be glad to point you in the right direction.

K
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Old 07-24-2008, 07:01 PM
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Default Re: Gel shift of ternary complexes.

Hey Kevin,
I appreciate the response. Yes, i have gathered a fair bit of reading material. But trying to optimize the conditions to visualise the shift is turning out to be a pain. Im trying to set up in vitro transcription reactions where my labeled DNA has a specific promoter element and is 200 bp in length. My crude extracts have the polymerase components and my binding conditions supply the NTPs and MgCl2 requirements. So i expect when i run on a 4% (79:1 or 39:1) polyacrylamide gel to see my transcription complex as a shifted complex. But i dont. So i dont know if my gel is run under conditions which destabilizes my complex.
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Old 07-24-2008, 08:31 PM
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Default Re: Gel shift of ternary complexes.

Thanks for the details.

One question about the protocol is the inclusion of the NTPs. It's been a while since I had to think about transcriptional initiation, but do your reactions also contain all the components of the holoenzyme that allow elongation? If so, the plymerase can just run right off the promoter? You won't see much of a shift if you don't get the enzyme bound and also stalled.


Anyway, Polymerase binding reaction conditions should be fairly common. I never worked with that one so I can't really tell you much other than to take look a several of the better papers (good data and detailed methods sections) so you can see what they did. When comparing the better methods sections you'll find common buffer components (such as the MgCl2), but also look to see if they are using DTT (many nuclear proteins like a reducing environment and often more is better ie >1mM DTT) and the incubation time/temperatures.

Are they using poly dI-dC or some of the analog. Salmon sperm is going to be a bad choice for polymerase.

Assuming the binding conditions are optimal, see if you can find out the strenght of the TBE buffer they are using to run the gel. Typically the gel is cast with 1X and then run is done with 0.5X, but it could be 0.25x. You want to make sure the ionic strength is not too high in the run buffer otherwise it will salt/knock the proteins off of each other and the DNA. Therefore, you'll want to pre-run the gel for about a hour to get the running buffer down into the gel and the 1X TBE out. Be sure to do the run in the cold room so the heat doesn't knock the protein off either. If you can afford them and have the compatible gel box, get the Invitrogen 5% DNA retardation gels - they have always done well for me.

200 bp is good and long. Are you seeing any non-specific bands? If not, try something like a generic Hela cell nuclear extract as a control so that you can at least visualize a shift even if it is not relevant.

Also, how are you detecting the shift? 32p, fluorescence or chemiluminescence?
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