6 replies to this topic

[Stef]

Hi all,

I am pretty new to the EMSA and Supershift experiments and i have few questions

I am looking at the gel shift of two oligonucleotides of 31bp that differ only on one base G vs A

I am able to have a very nice gel shift with the variant A, it can be displaced by A but not G allele.
The motif involved is a putative binding sequence for COUP-TF and/or PPAR gamma.

I tried PPARgamma and COUP-TF antibody (from SCBT) and i am only able to reduce the intensity of the band.
The PPARgamma antibody should not interfere with DNA binding as it's raised against the C terminus whereas the DNA binding is the N terminus domain

Attached is one example of my EMSA:
I am using Pierce Biotin EMSA kit and incubate antibodies 10min RT and then 20min RT for the EMSA (I use dIdC except for the last 4 lanes)





Lane
1: A biotin + G unlabelled + extract
2: A biotin + extract
3: A biotin + PPARg AB + extract
4: A biotin + Coup-TF AB + extract
5: A biotin + FoxD1 AB (should be the non specific control) + extract
6: A biotin + extract
7: A biotin + extract (Salmon sperm as non specific competitor)
8: A biotin + A unlabelled + extract
9: A biotin + G unlabelled + extract
10: A biotin alone

What can people can tell about my EMSA and "supershift"?
The top band seems to be a protein in my extract recognized by the streptavidin system as it appears also when I do a control protein alone.

Can i conclude only based on the decrease of the signal?


Thank you all for your help
PS: today i try first EMSA and then add antibodies

[aimikins]

hmm, it's a sticky thing to declare a SS based on signal reduction. although it makes sense and everything, it's not necessarily conclusive and can be hard to get past reviewers. it really is best to see if you can make that band happen.

changing the order of your reactions is good; you can also vary incubation time and temperature when looking for the supershift.

another thing is to tinker with the buffer; I wasn't able to find a band when looking for an SS until I fiddled around with the incubation, pre-ran the gel, and added BSA to the incubation buffer.  I just kept altering conditions until the band showed up.

good luck; it can be time-consuming but very rewarding

[Stef]

First, Thank you for the tips.

Secondly, I am still wondering what kind of supershift I am looking for..

My complex appears to run at 130-140kD, so if everything happens accorded to plans, i should had the mass of the antibody so roughly speaking around the same size 150kD which make the "super"complex at ~300kD.
Am i correct on this one?

Here is the latest EMSA:
20min RT EMSA,
10min RT antibody no BSA.




Is it possible that the faint band on top of my shift (as it appears like a doublet on lanes 3 and 4) is the supershift?
I would have expected a bigger size difference...

As suggested, i will try to change the conditions like time of incubation, and try with BSA. (which concentration should i try?)

I am also planning on trying running on a 4-20% gradient gel to get better resolution on the high MW without having the free oligos to run out of the gel.
And maybe try a hypershift with anti mouse antibody to supershift that putative supershifted band...

Are people that do EMSA still use big gel or most people moved to small gel (i use 6cmx8cm biorad miniprotean gel right now)
Also what range of TBE are people using (right now i use 0.5X)? because i will have to equilibrate at 0.5X the gradient gel from invitrogen from 1X to 0.5X TBE.

Thank you again

[aimikins]

I used big agarose gels, so I can't help you there.  I do believe Ab1 has a supershift. ideally that band would be a little more clean, but I think it's there!

as far as BSA, I did a range from 0.1% up to 6% and picked the one that gave the best bands.  you might have to experiment. BSA is supposed to sort of help stabilize protein complexes and I think that's why it can help.

[Stef]

I am glad that you believe in that supershift, i am myself pretty doubtful about it. (are we talking about the same band?: the small faint band a little bit higher than the lower migrating shift?)

I just checked for running the EMSA on a gradient gel but it will probably not help me (it might only give better resolution over 200kD but stack more the region of interest).
I guess the "only" way to get better resolution on the high molecular weight is to run a longer gel.

OR:
Does anyone tried or have heard of discontinuous gradient gel for an EMSA ?

I mean, almost all of the gel being useless, i am thinking doing a small high concentration acrylamide on the bottom to keep the free oligos to run out of the gel (15 to 20%) and the upper gel at 5% or even 4% and run the EMSA with a MW that will allow me to stack the low MW in the 10-20% and have good resolution for my ~140kD complex in the upper part.
It would be like crunching the half bottom part on the gel in 1cm and increase the resolution of the top half part.

Do you see any problem getting that type of experiment published?


EMSA seems pretty easy on paper but irl, so much conditions seems to be able to stop you from seeing a shift or a SS that i am in awe in some of the CREB EMSAs....

PS: i am also thinking adding a stacking part because if the SS complex is really >300 kD it might not get into the 5% gel....

[aimikins]

bigger gel, run it more slowly - I wouldn't try to make a figure out of a gel in which the bottom has run off, but that's up to you and where you want to publish.

[Stef]

No Aimikins,

I was thinking using a 20% acrylamide on the last 1cm of the gel to capture the free oligos and allow me to run the gel longer.
like this:

| U U U U U U U U |
|                         |
|                         |
|         4 to 5%     |
|                         |
|                         |
|______________|
|         20%         |
|______________|


It's kinda shrinking the bottom gel and make a longer run without having the free probe to run out of the gel. (right now i am using only the first quarter of the gel as i have no information in the bottom except the free oligos)

Thank you for the tips, i will try messing up with the parameters.
On the side i am also trying a DNA binding affinity column with my oligo to "purify" the complex and send it to LC-MS/MS

Edited by Stef, 20 May 2009 - 08:26 AM.