EMSA problems - weird film results (Sep/17/2008 )

Hi all!

I'm using the EMSA Kit from Pierce to carry out my EMSA experiments.

I've been consistently getting 2 rows of 'white spots' in my gel photo. Please see the image attached.
Has anyone encountered this problem before?

I would really appreciate if someone can help me out here. New to this technique.
And, I've been unable to get any bands. My samples are freshly prepared (20ug) and I follow the protocol from Pierce very closely. I'm unable to figure out why.

Would be really thankful for some help here.
Thanks alot!!

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Hi,

I can't see a photo. What kind of extracts are you using?? are they nuclear??

Sara

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Oh, here it is.




By the way, I'm using nuclear protein.

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If this were a western, this is what a reverse blot would look like. Usually it happens when you have too much protein, or use an inappropriate antibody dilution or a stronger detection method than necessary. I'm not sure what you are detecting here though with the EMSA kit - could you elaborate on the procedure?

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Hi,

I'm trying to detect binding of HIF (hypoxia inducible factor, a transcription factor) to the hypoxia response element.

EMSA was done using the Pierce Lightshift EMSA kit. Basically, 20ug of nuclear protein was used in each sample.
The sample mix was prepared by addition of the following in order:
ddH2O, binding buffer, 50% glycerol, MgCl2, KCl, poly (dl-dC), NP-40, BSA, DTT, EDTA. (bold items were pre-prepared and added to each tube as a Master Mix), nuclear extract, labeled probe.

The mixture was then left at room temp for 20min to allow binding to occur.

Subsequently, the mixture was then loaded into a native gel ( that has been pre-run at 100V,1h) and ran at 100V, 1h.
Transfer was carried out at 380mA, 1h.
Cross-linking was then performed. The membrane was then allowed to dry before storing overnight.
The running and transfer buffer used is 0.5x TBE. ( fresh buffer for gel run-- buffer from pre-run NOT recycled)


The 2nd day, the membrane was then blocked and washed before ECL incubation and detection.
What could have gone wrong? Anyone has encountered similar problems?

Thanks in advance!

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I can tell you what I'm seeing, but why it's occurring, I don't know. The white bands are proteins from your protein extracts. The black bands at the bottom of the blot are your unbound label. If there is a shift occurring you probably can't see it because its being blocked by the white protein bands. Question is why are the proteins showing up, and that I'm not sure of (probably some combination of what I mentioned above).

Some questions, just based on what you said and from reading the protocol: 1. are you using Nylon membrane as suggested? I suppose you could also use nitrocellulose, but Nylon is probably better for binding DNA, and could be a source of background. 2. You said that you blocked and then washed - in the protocol, there's a step for adding the HRP conjugated streptavidin to the blocking buffer before washing. Did you do this? I'm guessing that you did, just want to be certain. One other question might be how you arrrived at using 20 ug of protein. I'm not sure if this is a typical amount to use or not. You might want to try to decrease the protein concentration, or use several protein concentrations to get a curve (where you gradually see a shifted band). In any case, if you have the control DNA and protein provided with the kit, then you should probably test the system to be sure that everything is working right.

I'm sure that someone who has done this assay before could give you better advice, but thought I that I would just give you my input (I'm bored).

good luck,
smu

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Hmmm..thinking about this some more. I suppose it's possible that the white bands could be your shifted DNA, but I would still lean toward it being nuclear proteins. To be sure, you could run out some of your protein without the addition of probe (all other conditions being the same). If you see the same thing, then you know that it's from your extract.

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It looks to me that you have 3 bands present, and a non-specific smear at the top and bottom of the gel. The 'white spots' are where there is no band, and these should be present at the top of your gel also. You only need around 3-5 ug extract, and this excess (20 ug) is causing non-specific interactions that are not able to be competed out by the dIdC.

Increasing the concentration of dIdC, lowering the conc of extract, or adding an excess of unlabelled random ds DNA competitor should remove background smears, and make these bands clearer.

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