I'm performing EMSA in order to determine the Kd between human TBP and different 26-bp doublestranded oligos containing TATA-boxes. However, I've struggled a lot to get any shifts even though identical oligos earlier have been proven to interact with TBP with high affinity. I use the Gelshift™ Chemiluminescent EMSA Kit(Active Motif), recombinant TBP(Active Motif) and oligos synthesized by IDT.
The binding reactions contain 20 mM HEPES-KOH (pH 7.6), 5 mM MgCl2, 70 mM KCl, 1 mM DTT, 100 mg/ml BSA, 0.01% NP-40, 5% glycerol. Interaction has also been tested in absence of KCl, DTT, BSA and with the binding buffer provided by the kit instead of the HEPES-KOH. I have used different concentrations of DNA (0.1-100 nM) combined with different concentrations of TBP (1.25-125 nM). The bindings reactions have been incubated in either 30 min or 3 h at RT (TBP dimers has a quite slow disassociation constant and, hence the long incubation time was tested) before loading.
The gels used have either been 4% or 6% continuous native PAGE run in either Tris-glycine(1x) or TBE(1x and 0.5x). Buffers are not pH-adjusted. I run the gels in fresh buffer at 4°C with a constant voltage of 175 V.
I have had some initial problems with oligo hybridization and, therefore I run all my experiments with a ssDNA control to rule out that lacking oligo hybridization is the problem.
qPCR have been performed on anti-TBP immunoprecipitated sonicated genomic DNA and the regions containing the specific TATA-boxes of interest were amplified compared to control regions, so it seems like the TBP still have sufficient ability to bind the TATA boxes (at least when I'm performing immunprecipitation).
TBP has a quite high pI (9.8) so I've suspected that the net positive charge of TBP per se should be the problem. However, when bound to a 26 bp oligo the complex should have a net negative charge.
Does anyone have any suggestions for how to solve the problem?
Hello, Why dont you try to do it with radioactivity? it is much sensible that chemioluminiscence. Do you know is the protein is active? Have you tried to use a bigger quantity of protein? Maybe the Kd is very high. Other thing is that is the DNA es so short maybe you need to increase the acrilamide concentration to see the sifht. Good luck.
Hi Steffan,
Reading your detailed description, I come to the conclusion that everything is o.k. and it should work. My experience is only radioactive EMSA and therefore I have no experience with biotinylated oligos.
You refer to possible problems with the hybridization. Annealing usually works well, however the annealed oligos should be stored in the presence of salt (app. 100 mM). Your annealed oligo should run slower than your ssDNA control.
Did you try an EMSA without detergent. Some interactions are sensitive to NP-40.
Do you use a loading dye? It may also affect binding.
Good luck!
Hello Steffan
From our experience, the radioactive protocol for EMSA is more sensitive. I have performed EMSA using alpha32P-dCTP for radiolabelling double-stranded oligonucleotides. The reaction mixtures (without the loading dye) were subjected to electrophoresis in 5-6% polyacrylamide gels (Accugel 29:1 sequencing grade) prepared in 0.25x TBE buffer. Loading dye can be added to an empty well at the end. Description of the protocol appears in our papers (A&R 2005, 52(9): 2906-2916; A&R 2008, 58(1): 273-283). All the best with EMSA.
Hi Steffan, How does the wells with protein look? Do they appear darker than the oligo only well?
We have done TBP/TATA EMSA quite successfully. I did it many years back and you can see them in the attached publications.
Article TFIIA abrogates the effects of inhibition by HMGB1 but not E...
Article Influence of HMG-1 and Adenovirus Oncoprotein E1A on Early S...
Thanks for the answers!
The main reason for using this kit is that one that is available in the lab. However, to my experience it should be sensitive enough to detect the expected shifts. Elena, do you have experience with both kinds of EMSA? I usually load 1 nM oligo but I can still clearly see the 0.1 nM band when it is included in my experiments. Even when I overexpose the film, I see no specific shifts. The combined qPCR and immunoprecipitation should have confirmed the activity for the protein when using the same components in the binding reactions. However, I have not been able to prove that the protein (same stock) is active when I use it for EMSA.
The oligos are stored in 50 mM NaCl, 0.1 mM EDTA and 10 mM Tris, pH 8 and are rehybridized before each experiment. The ssDNA migrates longer than the dsDNA every time I run with TBE buffer. However, when I run with 1x Tris-Glycine ssDNA and dsDNA migrate the same distance. Of some unknown reason, these conditions influences either the migration distance of the oligos or the stability of the duplexes. I have not tried without NP-40 yet. I'll give it a try. I use a 5x loading buffer provided by the kit but don't know the specific components of the loading buffer.
I have never been used this type of EMSA only with radiactivity. You said that with 0,1 nM of probe you can see it, but what you see is free DNA which is maybe the most abundant.
Anyway my EMSA are different I use 250 bp dsDNA fragment. And for this size of DNA I use acrilimide 5%. Maybe you should try to increase the acrilamide concentration because I suppose that your fragment is very small. Sometimes changing concentraction it works.
About the loading buffer I use the same that I have for the agarose gel (6x) and I dont have any problem. If you have NaCl try to reduce it at maximun because affect to the binding.
What I meant was, that I'm able to detect even quite low concentrations of probe. When I run the experiments with high concentrations of either probe or TBP, I should be able to detect even a very small fraction that was shifted.
Atreyi, when I perform EMSA with the chemiluminescent kit, the wells are not visualzed in the same way as I guess they are in a radioactive assay. However, it does not seems like any complexes are stuck in the well. Do you remember if you used 0.35X TBE, 0.05% NP-40 for all experiments or did you try different buffer conditions such as adjusted pH, higher TBE concentration, without NP-40, etc.?
Hi Steffan
Not sure about your specific system, but I have used biotinylated/TBE system in the past successfully. Doubt strongly that your problem has to do with your oligo. Now, never worked with TBP though. Clearly would try sticking to the published high-affinity binding protocol you allude to as this is likely where the problem resides. One parameter that you may consider is to try your shift in the absence of any loading dye which could interfere with binding (it did in those days on our particular target). As mentioned above, detergents are also major game changers. Would try binding AND run under pure 0.5 X TBE (very permissive conditions) and validate binding with a supershift. What about your TBP...could it be inactive? What's your source? Any known PTM that could inhibit your binding? Does it look good on a SDS-PAGE gel?
Good luck!
Sebastien, did you experience that the loading buffer directly interfered and reduced protein:DNA association? I have not considered at all that the loading buffer could be a problem.
I'm quite sure that the TBP is active. As mentioned earlier, the TBP has the ability to bind to the specific TATA-boxes in DNA immunoprecipitation with sonicated gDNA. Regions containing the speific TATA-boxes are amplified compared to regions without TATA-boxes when I perform qPCR on the immunoprecipitated DNA.
I have not excluded the possibility that some PTM may be inhibit the binding. The protein is expressed in E.Coli with a N-terminal His-Tag. When I run the protein on SDS page it has the expected size and there is no other bands.
Hi Steffan,
We always used the 0.35x formulation. The protocol was optimized at the Pugh Lab. We have never had any problem with the binding - sometimes non specific bands with higher mobility would appear though. Can you add some activators, co-activators in your reaction that are known enhancer of TBP/TATA binding as a positive control?
I would prefer to investigate the binding in absence of any other proteins, activators etc, but I'll still consider use it until I know what I'm doing wrong with my experiments. Do you have any specific activators in mind?
The supplier protocol you are using is quite convoluted, hence more possible issues. Why did you chose the biotin-SA system? Does TBP even bind to nylon membranes well? Wondering if your problem isn't simply that your protein doesn't bind efficiently to nylon and/or that the biotin is buried inside TBP and not accessible to SA
You could try straight western on nylon (never tried myselft; or a control PVDF transfer) or even staining your gel with your favorite sensitive protein stain to make sure TBP enters your gel.
My experience was with non-transfer based system (radioactivity or fluorescent labeling of probe itself) which bypasses the need to expose your probe to SA. Not sure what your options are currently but I prefer the devil I know on this one and, if you have access to a fluorescent based imager (Typhoon, Odyssey etc..) I would switch to a fluorescently tagged probes...
Your control suggests that TBP isn' t the problem but you could still make sure that your recombinant protein looks good on gel by itself.
If you want to convince yourself you can (in your system) shift things, why don't your try nuclear extracts? They contain multiple proteins, including the abundant (in humans) Ku70/80 dimer that binds DNA ends very well and will shift in a heartbeat any oligo incubated with nuclear extracts.
Good luck!
Thanks for the detailed answer! I do unfortunately only have access to perform the chemiluminescent EMSA but it seems like most will recommend the radioactive or fluorescent assays. I guess, it should not be necessary for me to make sure that the TBP binds to the membrane since it is the oligos that are biotin-labelled and, hence I should only worry about the transfer of the oligos. Anyway, it is a good idea to do the staining of the gel and then get an estimate of if the TBP is able to enter the gel at all.
The TBP looks fine on a SDS page but unfortunately that does not provide any information about activity. Need to find a way to estimate the activity of the TBP instead of the qDIP but can't really figure out how to make that experiment.
The system is available to shift the probes. The kit provides nuclear extracts + biotin-labelled oligos and these shifts run good every time. Actually, my own oligo designs was also able to cause shift when added together with the nuclear extract. However, I don't know if that shift is caused by binding to Ku70/80 dimers, TBP or another DNA binding protein.
I agree in principle with your view, but if TBP sequesters the oligo, the oligo might be not be able to interact with the membrane...pure speculation mind you!
Looks like you did a fine job with your controls. I assume you have a terminal biotin on your oligo, removed from your actual binding site. We've had good experience with IDT's oligos (fluorescence and biotin) but issues with oligo synthesis remain to be considered (we've had some issues before with a different company).
You could consider a streptavidin pulldown approach of your preincubated oligo + TBP...does TBP come down ( assessed by western blot)? Could solve your potential charge quandary and deal with PAGE related issues.
Good luck!
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