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I'm in EMSA Pain!

I'm in EMSA Pain! - Protocols and Methods Forum

I'm in EMSA Pain! - Post Any Protocol, Method, Technique, Procedure or Tips / Troubleshooting for any Molecular Biology Technique.


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  #1  
Old 07-13-2006, 02:14 PM
Pipette Filler
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Question I'm in EMSA Pain!



Hello to all the scientists on this molecular biology forum!

I have a problem that I hope someone out there can help me with! I am trying to carry out a series of EMSAs to characterise the binding of a transcription factor to the promoter region of my target gene. I have designed PCR primers to amplify a product of about 200-300bp which I then gel extracted, purified and end-labelled with T4-PNK. I have done the same for a positive control (guaranteed to work) and a -ve control. I have purified protein of fairly high concentration that is ready to go. Up to this point, all is well.

Now when I carry out my band shift, the positive control shifts and the negative control does not, as expected . My test fragment however, shows up as a double band when exposed (even in lanes where I have not added any protein - meaning that it there is something wrong with that DNA fragment). I first thought that this may be due to two unique conformations of my probe fragment, meaning that one conformation runs slower than the other, but when I redesigned my primers so that they do not amplify across a region with runs of A/T repeats (known to induce DNA bending) I get the same result.
Has anyone else experienced a PCR product probe that runs in two bands on the polyacrylamide gel-shift gel even though it was purified as one band following agarose gel electrophoresis?

I could really do with some help here from an EMSA expert. I can provide a picture of my latest developed film upon request (I don't want to jam up the forum on my very first post)

Many thanks to anyone who will help me!

Canalba, UK
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  #2  
Old 07-13-2006, 05:01 PM
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Default Re: I'm in EMSA Pain!

Hello Canalba,

I have done EMSAs almost all my research life! Please post the EMSA film if you would like, I could certainly look at it.

I have done many EMSAs and all I can say is I have had this problem before.
THis can be caused by the following:

1) are you using DNases at any step during the EMSA or DNAse inhibitors? these can bind to your DNA sequence and cause shifts on the EMSA

2) your DNA can alter in its running due to changes in salt / buffer / purification and other steps you do after the running of the agarose gel to check it

3) this is usually considered normal. what is the size of this shift? is it very low on the gel? if it is high, you may have some troubles explaining that as it may approach the range of some of your complexes.

Please post a reply / or a film so we can see how you are doing!

Cheers!
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Old 07-13-2006, 06:05 PM
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Red face Re: I'm in EMSA Pain!

Hello!

THANK YOU for replying! I was getting a bit worried because as usual, everything is on a deadline! Anyway, ~I wanted to answer your questions real quick so you can get a better handle on what i'm doing.

>1) are you using DNases at any step during the EMSA or DNAse inhibitors? these can bind to your DNA sequence and cause shifts on the EMSA
No...I'm not using DNases or DNase inhibitors, however I do use a protease inhibitor cocktail which I add to all my samples prior to incubation/running. It may be this, but the double probe band does not appear for all my probes, only 2 out of 5 of them, but the two are the key fragments (as usual)

2) your DNA can alter in its running due to changes in salt / buffer / purification and other steps you do after the running of the agarose gel to check it
Following agarose gel running, I perform a normal gel extraction using the qiagen kit, followed by elution in water. Again, all my samples have been treated in the same way, so unless it is a phenomenon tht can happen to some bits of DNA and not others, then I don't think it could be this....hmmm...

3) this is usually considered normal. what is the size of this shift? is it very low on the gel? if it is high, you may have some troubles explaining that as it may approach the range of some of your complexes.
This is where we get to the irritating part - the shift is round about where my complexes are likely to appear....and I believe that they may be masking the complexes

Now I have attached a couple of images for you to see what I have been doing. The first file (EMSA001 s) shows the gel with my positive control DNA to show you the sort of shift I'm expecting (Lanes 1-7). I think the legend on the bottom explains this too. In the same picture, you'll see a promoter region fragment that I know does not have a binding site and therefore does not shift, as expected (Lanes8-10) - the last three lanes are a negative control not expected to bind this DNA at all hence no shift (Lanes 11-13). Good Job so far, right?

The second film I have to show you (EMSA002 s) is of a more dilute range of protein concentrations for my positive control (Lanes 1-7) as I was playing around with protein concentrations to try and find the optimum range. Lanes 8-10 contain the same DNA probe from lanes 8-10 in EMSA001s, which confirms that result. Lanes 11-13 are of another promoter region fragment, this time closer to the start codon of my target gene which run in this double band effect that I'm struggling with. I know that this film is slightly overexposed but I think that the probe level drops ever so slightly across lanes 11-13 showing that there may be binding, but I can't visualise it due to the secondary bands. Incidentally, the DNA probe in Lanes 8-10 is very close in molecular weight to that of the probe in Lanes 11-13, so I believe it is the lower band that I am really interested in?

I'm really sorry for the text overkill in this post, but it's so nice to get a sympathetic, knowledegable ear that I think I went off on one a little bit.

Well there it is...I don't know what to do short of ordering oligos that cover the putative binding site and starting again (not my favourite option)...

What do you think??

I await your comment, but will probably have to post a reply tomorrow as I need to leave the lab for cleaning...I'll try to log on later to check in any case.

Thank you once again for your time...

Canalba, UK

EMSA002 s.jpg

EMSA001 s.jpg
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Old 07-13-2006, 06:07 PM
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Default Re: I'm in EMSA Pain!

OoOops..In the paragraphs earlier, EMSA001s refers to the second image and EMSA002s refers to the first image as you see it in the post.
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Old 07-14-2006, 01:19 AM
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Default Re: I'm in EMSA Pain!

Hmm... beautiful EMSAs by the way!

I see that they are great bands for your complex and they increase with increasing protein concentration. Thats good because its dose dependent. I see your free probe is decrease as the protein increases because its making complexes. Great.

Only thing I see that may be a problem is 2 things.

1) what is the size of your HPR2 DNA it seems very big. Big DNA tends to have problems as it can be nicked, supercoiled, and normal. This gives several bands on a gel (even agarose) and would do the same on an EMSA. This is one possibility, as I see two more higher bands. One thing you could do as you mentioned I guess is try a smaller probe to prevent the free probes being visualized "on top" of your complex bands. However, it seems you may be using much higher pmoles of hot probe for your negative control giving you those secondary bands. try to use the same pmoles (calculate the pmoles for the bigger DNA - you should be using less probe for your bigger DNA probe). This could solve your problem!

2) also make sure you use cold - DNA promoter to try and out compete your complex and see if it is in fact specific (you may have done this already)

more coming soon

Last edited by admin; 07-14-2006 at 01:32 AM.
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Old 07-14-2006, 08:52 AM
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Default Re: I'm in EMSA Pain!

Thank you very much for the advice, admin!

I agree with your assessment about the HPR2 DNA. The probe is about 330bp whereas the positive ctrl probe (PHR1 promoter DNA) is about 180bp, sothere may be an issue there. I have ordered and repeated the EMSA with smaller probe fragments (~180-200) from my target gene promoter, but these too have the same double band effect.

I have not yet tried the cold promoter DNA vs positive control titration/competition experiment but I think that it will soon have to be done. I just wanted to try everything I could to get a proper band shift with my promoters themselves, as I have previously been advised that this is stronger data? Or is it just as convincing with the competition study? I wonder if the protein will stick to any DNA if there is enough of it around?

I'm also considering ordering oligos (~20-30mer) encoding the sequence across the potential binding site in the promoter region and using these as probes. This will give rise to afew questions...
1)Could you give me any advice about this type of experiment?
2)i.e. Do you think it may get rid of the multiple conformation/band effect because they are so small?
3)Have you ever done this and has it worked for you?
4)Can I label it in the same way, and how do I purify probe of only 30 nucleotides? (most commerical DNA purification systems seem to cut off around 15-200bp...
I strongly feel that these binding sites will turn out to be much weaker than my positive control so the range of protein concentrations necessary may be higher to see the same shift...?

Looking forward to your next post...

Thanks again!

Canalba, UK
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  #7  
Old 07-18-2006, 03:59 AM
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Default Re: I'm in EMSA Pain!

Hey again!
you could easily order a few oligos or create restriction site digestion fragments and label them... use these then as probes after purification in case of restriction enzyme cuts or just anneal the two oligos together and then label them

yes smaller probes should remove the higher bands you see on EMSA. although I mentioned you should use less of it ( I think you are using too much pmoles maybe of the larger probe)

to purify use G-25 sephadex for small probes although you will not need to purify oligos as they are already pure

Cheers!
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Old 03-21-2007, 04:58 AM
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Default Re: I'm in EMSA Pain!

Thanks for posting this problem and possible solutions. If I may, how things are progressing with your assays?

I have been having similar problems with equal intensity secondary bands in negative controls. I went through addition of each binding buffer component to identify a potential contaminant with binding capability when dna and water exhibited the extra band. The DNA is relatively long (278bp) and AT rich, so i suspect that it is due to bending however the band is running near my bound 26 kD protein shift (not exact). Do we expect a bent DNA to shift this much? Can we eliminate this (not using shorter fragments is not really an option now)? Why doesn't all of the DNA run the same as the 'bent' DNA?

Thanks in advance!!!
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