DNA Binding

DNA Binding – Science topic

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Debanjan Bhattacharya
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Hi all, Recently I've been having issues with my SDS-PAGE gels not running properly. The samples don't run in a straight line as they should, but instead the lanes in the middle of the gel migrate way behind the lanes at the edges of the gel, leading to a dye front in the shape of a frowny-face. In addition, the lanes in the middle of the gel compress and become narrower, while the lanes at the edges of the gel expand and become much wider than the width of a lane. Does anyone know what might be going wrong? The gel rig itself is not the issue since another lab member has been using it for... more
This is most likely due to either of two situations below: Either buffer is level is decreasing in the gel cassette due to a minor leak. Check that. If buffer leakage is not there you may want to try a fresh SDS running buffer. There my be bubbles in some parts of the gel (some visible in the lower part). Remove bubbles before loading samples and running the gel.
Shamsher Singh Kanwar
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Hi all, I am using an ELISA method for detecting DNA and have established that Protamine Sulfate looks best for binding DNA to the plate. Does anyone have any suggestions for starting concentrations to bind DNA or if coating and leaving overnight has any benefits as opposed to a shorter coating time? Thanks!
Protamine otherwise has great affinity for binding for IgM class of antibody. Simply dissolve DNA in carbonate bicarbonate buffer of pH 9.3, and use it for coating on to Wells of ELISA plate.
Ad Ad
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I'm running an EMSA and I'm finding that adding additional protein causes the free probe band to start to disappear instead of shift up into a new band. It seemed to work before so I don't really understand what the problem might be. I've attached a picture of three gels. Each gel is basically probe 1 being exposed to increasing concentration for 7 lanes and probe 2 being exposed to increasing concentration of protein for 7 lanes. Probe 1 shifts into a faint blurry band but probe 2 just seems to shift slightly and mostly just disappear. What could be the problem? For the mix I'm loading... more
I appear to have fixed the immediate problem by upping the protein concentration. Thanks for your help.
Sondavid Nandanwar
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i synthesized organometallic compounds and i want to check DNA binding. For that i try to prepare the solution of organometallic compounds in TRIS buffer. But soltion get precipitated. Now i want to know any alternative to TRIS buffer.
Sondavid Nandanwar
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I synthesized organometallic compounds. I want to do DNA binding study of these compounds using Gel electrophresis experiment. Can i use CT DNA for this study? Please help me.
CT DNA is calf thymus DNA.
Umayya Yasmin
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i synthesized tetraaniline, polymerize with fumaryl chloride and Polyethylene Glycol m.w=4000. now i want to study it's applications in various fields i.e. DNA binding,DPPH, CV etc... kindly help me to solve this problem and suggest me what more activities i can further perform.
thanks to all
Eunice Lim
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I'm using Zymogen Maxiprep to extract my plasmid from NEB Stable competent cells. For some reason, when I add DNA binding buffer to the lysate (cells lysed, neutralized, isolated from precipitate), the lysate turns cloudy, pinkish, and forms oil droplets! In previous successful maxipreps when I added DNA binding buffer, the lysate remained clear and yellow. Note: I don't think this is a problem with having too many cells because I've used 25 mL of culture and still got the same weird reaction.
Hi Kimberly, Extending the lysis time to 5 minutes (which I notice is the instructed amount of time for maxipreps from other kits) worked and the solution remained clear after adding DNA binding buffer - no oil droplets, cloudiness, or color change. Thanks for your input.
Jeffrey Nicholas Mcknight
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We want to use a chimera of DNA binding protein (to a specific DNA sequence) and strepavidin. Do you know of any from the literature or personal experience?
Malcolm, while I have no experience with streptavidin-DBD fusions I do have experience creating a fusion between a chromatin remodeling protein (Chd1) and streptavidin. (if interested, see https://academic.oup.com/nar/article/41/3/1637/2903312) We needed to disrupt the tetramer interface to get this to work properly for our work (see http://www.jbc.org/content/280/24/23225.long). The monomeric streptavidin was better-behaved for our purposes. I don't imagine much difficulty creating a fusion to a DNA binding domain - do you have anything specific you want to know about such fusion...more
Mohammad Issawi
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In general DNA targeted chemotherapeutic drug, interact with DNA and inhibit the DNA function finally cause the cell death (both normal and cancer cell). To understand the DNA binding ability of the drug DNA binding studies have been carried out. But, In PDT, photosensitizer kill the cells in the presence of light. Then what is the meaning to study DNA binding affinity of the photosensitizer. Can't it inhibit the DNA function and kill the cell as normal DNA targeted chemotherapeutic drug?. ok if the photosensitizer shows weak interaction with DNA then it won't kill the cell via DNA... more
PS-DNA interaction has been studied due to PS toxicity (at high concentration) under dark conditions. In PDT (in vivo) TPPS and TMPyP porhyrins have been relocalized into the nucleus after irradiation. Then interaction with DNA may not always lead to cell death, it may induce mutations.
Subramaniyam Ravichandran
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Hi all, I am performing a Co-IP experiment where would like to check the binding of my protein to the DNA of interest. I kept a control where I incubated the DNA and antibody only with the protein A/G beads, after which I performed PCR to amplify the DNA of my interest. I am seeing the desired PCR bands even in the control. Is it possible that the protein A/G beads show some sort of nonspecific binding to my DNA of interest resulting in these bands? I found the following links mentioning nonspecific binding of DNA to Co-IP beads: Chromatin Immunoprecipitation to Analyze DNA Binding... more
Hi Pratibha, I have not added my protein to the mixture at all. The mixture only contains the beads, antibody, and DNA. The purpose is only to check whether the DNA can nonspecifically bind to the agarose beads. Thanks!
Geoff Margison
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This question is related to my Ph.D work
I can completely support Martin's suggestion. Sybr Gold is excellent for both SS and DS DNA. We are detecting less than 100fmoles of short (~15-mer) oligonucleotides in PAGE gels (although we do use a Molecular Dynamics phosphorimager to view the staining).
Mithun K Ghosh
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it may be H - bonding between metal complex and DNA
Tomas Koltai
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I am searching for a protein (or transcription factor) that can interact with a short DNA sequence (that is NOT promoter or some part of promoter for another gene) and this interaction results in producing signal that control the expression of another transcription factor. Can someone suggest such a regulatory network in plants, animals or other organisms?
As an example of what you are looking for: Sp1 (a transcription factor) interacts with the gene of HIF-1alfa (another transcription factor) that controls more than 100 genes.
Mamta Rani
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I found one protein which is having ABC transporter motif along wit Zn finger DNA binding domains, is there any study which shows that a transporter protein can work as a transcription factor.
Thanks
Ying-Xian Goh
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We all knew that antitoxins are highly specific for their associated toxins and cross-talk is similarly unlikely to occur in vivo (Ramage et al. 2009). However, if a toxin deletion mutant was constructed, where will the antitoxin bind to if there isn't any toxin? Will the antitoxin bind to the DNA and affect its replication since an antitoxin have a DNA binding domain? Many thanks in advance!
I see. I will try to delete the whole system in the future. Thanks!
Uttam Pal
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What is the different between them? and can we you UV-Visible machine as CD ? more information please.
OD is simply the optical density of a sample. It is basically the ratio of transmitted light (I) and incident light (I0) intensities, -log(I/I0). Whereas, CD is the difference between the absorptions of two different kind of (polarized) incident lights (left and right circularly polarized). Regarding measurement, it depends on the machine. Some machines may allow you to measure CD, OD, fluorescence etc. simultaneously.
Farah Deeba
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I have been making new protein preps for my EMSA experiments and have been getting enough protein, but when I verify DNA binding activity against my control probe I am not seeing any shifts. These were working just a month ago and now I can not seem to get any positive results. The protocol has not changed. Any help troubleshooting would be greatly appreciated.
Did you see any difference in probe intensity without protein or with protein? You can run Kit control reaction to see whether your apparatus is working fine or not.
Gautier Richard
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Can be possible the transcription factor's dna binding domain's binding to two or more different DNA sequence in different(or also same) enhancers? Or there is no exception, it must be specific for dna sequence like CAATTAGTCA... for example my book says; ''MITF's binding domain can attach just the one dna sequence in the different specific enhancers regions. Is there any different possibility?
Yes they can. Actually if you check Transcription Factor Binding Sites databases, you can often find DNA Binding Transcription Factors that bind multiple DNA motifs (so at least two different DNA sequences). For example the tramtrack protein in Drosophila melanogaster: http://mccb.umassmed.edu/ffs/TFdetails.php?FlybaseID=FBgn0003870
Adam B Shapiro
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Methylene blue is widely used as powerful photosensitizer in PDT and antimicrobial photodynamic therapy. In the other hand, methylene blue is commonly used for DNA staining and sometimes it shows dark cytotoxicity/genotoxicity. How can a DNA binder with potential toxicity deal with such selective photodynamic modalities??
Dose, not dosimetry.
Manjunatha Kogenaru
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Can you list some synthetic transcription factors that are common and effective? i.e DNA binding domain(recognize and bind certain DNA) + transcription activating domain(drive transcription)
Following are the most popular programmable DNA-binding domains that allow you to design a custom transcription factors, which can bind to your desired DNA sequence. These can then be used as a transcriptional activators/repressors/nucleases/epigenetic modifiers based on the effector module fused to the programmed DNA-binding module: 1. CRISPR-Cas9/dCas9 2. Transcription Activator-Like Effectors (TALEs) 3. Zinc-fingers The above order ranks the platforms from easy to difficult in designing. Hope this is useful to you. All the best, Manjunatha
Sherwin Lehrer
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I'm studying DNA binding by a protein. The DNA has 5'-Fluorescein tag. On protein binding, the anisotropy decreases unlike expected. I used excitation 495nm and emission 520nm with slit width 5nm on a Horiba Fluorolog spectrometer. I have varied integration time from 1 to 3s. Can anyone explain why I don't see an increase? Is it possible if my fluorophore is unstable?
The simplest experiment is to measure the fluorescence intensity because intensity is proportional to lifetime, with and without the perturbation, i.e., the protein, under identical conditions, when there is no instrumental reasons for intensity changes, like light scattering. In that case any change would be dependent on lifetime changes . A more direct experiment is to measure the lifetime change which is not affected by light scattering. From the lifetime change one could estimate the change in anisotropy for no change in flexibility from the Perrin equation: ro/r = 1 + tau/theta,...more
Ranjan Kumar Mohapatra
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Benzaldehyde derivatives showing DNA binding nature.
Dear Prof. Chen, I wish some benzaldehyde derivatives showing DNA binding/cleavage activity. Only the organic compounds but not with metal ions. Well, thank you for providing the link. Sincerely, Dr. R. K. Mohapatra
Verruchi Gupta
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Is the DNA binding chemistry between DNA and column provided with the kit same in case of PCR purification kit and gel extraction kit?
I have one question...I have done pcr and it shows band on the gel but after using gel extraction kit no DNA on gel please tell me how would I elute the DNA from agarose gel??
Ireneusz Stolarek
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I have this question because when you use softwares to predict transcription factor binding site they give you binding sites on both strands so which strand should we consider, the coding strand or both?
For the reporting the positions you should use the strand that the software has returned, as that is the place where the transcription factor is predicted to have a binding sequence present. Apart from things, that we don't know the exact occupancy state of the DNA region at and near this sequence, the transcription facotr is looking for that sequence motif to bind to, and this or that strand of DNA has that sequence motif, so unless you are working on some 3d genome modelling or advanced molecular biology question regarding transcription factors don't overthink it and report the motif,...more
A. Javadmanesh
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To perform HRM analysis for genotyping on CFX96 machine for the first time, BioRad recommends to use HRM calibration kit along with Precision melt analysis software. I am just wondering whether simply using any good quality DNA binding dye along with standard PCR reagents will work or not?
Hello all. For some reasons I have lost the calibration file! I can see my previous HRM experiments data on the precision melt software but when I want to import any new data, problem pops up! The error message is there is no calibration data. Can anyone borrow my a calibration file just to see if it can help? My machine is the BioRad C1000. Thank you.
Aziz Khan
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I want to find the binding domain of NF-kB
I'll recommend to use JASPAR too http://jaspar.genereg.net
Paul Rutland
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Would the fallowing be possible? 5'ATGGCATT3' 5'TACCGTAA3' Is this sort of conformation sterically impossible? or is it possible but just not energetically favorable? Any reference material would be really appreciated! Thanks!
Thanks for that Vlad. The authors of the paper that I sent you are on RG and show the practical application of PCR amplifying a reproducible but "incorrect" amplimer. G quadruplex alsocomes under the unusual base pairing possibilities. It looks like many structures may be possible if the energy can be supplied to get over apparently unlikely pairings. Have fun with it Paul
Ruta Gerasimaite
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Hello I have performed EMSA and got reasonable experimental image. So I want to draw binding curve and Kd value out of it, but I don't know how to do. I used P-32 labelled DNA and got phosphorimage. so I tried quantify free-DNA and protein bound-DNA bands using multigauge 3.0(Fuji film). there's no problem in quantifying free-DNA bands, but protein bound-DNA bands are smeared, or sometimes gives multiple shifted bands, giving unreasonable values. can I just use free-DNA bands only? Anyway I quantified free-DNA bands and then plotted fractions of free-DNA. and then I wanted draw the DNA... more
As already suggested, you can use bound DNA value to compute Kd using SigmaPlot or similar software. However, from the gels above it looks that your situation is more complex than single-site binding. There clearly two complexes with different electrophoretic mobility and affinity. It is possible that two protein molecules may bind DNA substrate. If it makes sense, you should get two Kd values. Such software as Dynafit allows you to fit your data to alternative mechanisms.
Neville S Ng
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How does the DNA binding property of the complexes impact antiproliferative activity or cytotoxicity
When the words small molecule, DNA binding, and antiproliferative cytotoxicity come together, there isn't room for a whole lot of variation in what happens in the most general sense. A lot of cell death, cell cycle arrest, apoptosis, necrosis, or senescence. However specifically, investigations often consider DNA minor groove/major groove/intercalative binding, or ss/ds DNA cleavage. DNA cleavage is often considered for copper complexes due to relevance to reactive oxygen species generation. Mitochondrial not just nuclear DNA has also been considered to be a potential major target, which...more
Paul Rutland
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I just know from Southern Blotting, that ssDNA binds to it
Double stranded dna binds to both nitrocellulose and nylon membranes and in Southern blotting the transfer of dna from the gel is double stranded dna having just been cut by a restriction enzyme. This comes up as a new question but from 4 years ago..can this be right?
Pavla Stojkova
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Hi, could you give me an advice about chromatin immunoprecipitation in bacteria? Is it possible to use DNase for removal nonbinding part of DNA? I mean this: protein will be bind on part of digested DNA, you will use DNase for removal part of DNA, which doesn´t bind my protein. Theoretically, it will remain only concrete part of DNA that bind my protein, because the protein protect phosphodiester bound. What do you think?
Thank you very much :-) I will read up it.  btw, chromatin - you know what I mean :-)
Wajih Al-Soufi
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We are measuring the anisotropy of a small oligocationic molecule after each addition of a DNA solution. The titration profile fits rather well to a 1:1 binding mode (as we expected), but the first point (small molecule before addition of DNA) is too low. I believe that upon adding the first aliquot of DNA the nonspecific electrostatic interactions generate many low affinity complexes that capture all the DNA binding agent (so the second point goes up), and then upon adding more DNA the molecule can go into its proper binding site. Does it make any sense?. Attached the titration. First... more
Hi Eugenio, we could have a look at your data the next days!
Karen Huang
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I would like to see if Muc1 binding aptamer(5TR) selectively binds to MUC1-expressing tumor cells by flow cytometry, but I failed to see the signal shifting from my control group.  I guess my problems are in the washing processes or the binding conditions.I can see an obvious signal shifting (similar as the following picture presented by a paper)after incubating mcf-7 cells with FITC-labeled aptamer(5TR) by flow cytometry without washing steps.But! When I started adding the washing process as the protocol and papers told, the result showed no shifting comparing with my control group (no... more
Kalani Jayanetti
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Hi, I'm doing a MD study about DBD of Glucocorticoid receptor. Why do it have two Zn motifs ( protein chains)? Both have same function or different functions? What is the importance of having two chains?
Hi dominique, It's helpful.Thank you.
Sutirtha Kamal Sen
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In the presence of cyclic-AMP, the Catabolite Activator protein (CAP) binds to the major grooves of DNA via its DNA-binding domain (DBD). This feature enables the phenomenon of transcription. My question is, is there any particular sequence of base pairs that enable this binding? Is there any other specialty in the major groove to facilitate this binding?
David Nemazee
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Hi, I am working with <50 nt ssDNA oligo's that I have purchased. I would like to see these oligo's on a gel, however they are quite small so I must load the gel with lots of dye to see a faint band and this gives lots of background fluorescence. The dye I am using is RedSafe. I am looking for ways to see the bands without using DNA binding dyes. I have considered using radiolabelled oligos but my lab cannot work with radioactive substances. I know that IDT sells oligo's linked to fluorophores, are these suitable to be run on a gel and visualized? Thanks.
Hi Ranvir, Try using 10-20% acrylamide gel followed by staining with SYBR Green.
Cameron N Gundry
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I have made Real Time PCR master mix and added 1ul SYBR Green in each reaction, but i haven't any appropriate curve.This problem has happened several times in the past, even trying it by different master mix and different volume of SYBR Green.Thanks.
My first advice, check for product, as others said above.  All dyes inhibit PCR a little, or a lot. But SYBR Green inhibits PCR less than ethidium bromide does when considering molarity and fluorescence, so that's what started the SYBR Green revolution in qPCR--Invented by Carl Wittwer. It sounds like you could be ordering the dye SYBR Green concentrate http://www.lifetechnologies.com/order/catalog/product/S7563  and adding it to a home brew PCR mix. Back in the old days before there were SYBR Green master mixes, people used to do this all the time. Not any more. If this is what you're...more
Venkata Narasimha Kadali
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Please provide composition of DNA Binding Buffer.
Eman Alwattar
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what the type of binding of F.B with DNA (by groove or other) beside I want know the extension coefficient for Fuchsine basic and in which buffer because I try to determine the extinction coefficient more than one time and it gives me different slope why? is that related to the structure or  what exactly?
Adam B Shapiro
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I am interested in looking metabolite-protein interactions. Does anyone know if fluorescence resonance energy transfer (FRET) is applicable to this type of interaction or is exclusive to study protein-protein bindings?
FRET can be used for this application. You need a donor and acceptor, and they have to be close enough together in the bound state. If you are lucky, the metabolite can act as either the donor or acceptor due to its own spectral properties. If you are even luckier, the intrinsic tryptophan of the protein can be used as well (probably as the donor). If that isn't the case, you will have to introduce extrinsic labels by chemical means. Many fluorescent dyes are available commercially for this purpose. Sometimes, you can take advantage of naturally occurring Cys residues in the protein as a...more
Mohamed Ibrahim Khalil
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I am planning to test if my protein will bind to my test promoters through non-radioactive EMSA. In this case, I will just purify the proteins and synthesize the test promoters and incubate them in a phosphate buffer/Tris buffer in the absence  or presence of my test substrate. The samples will be stained with dye and will be loaded onto a 1.5% agarose gel with ETBR. Is this advisable? In addition, what is the difference between radioactive and non-radioactive EMSA in terms of results? If you can help me, it will be a great help to my experiment. 
I used the lightShift Chemiluminescent EMSA before but unfortunately it did not work as expected and heard from others. The size of the biotin molecule is very large compared to P32 which affect the binding of the proteins to the DNA probe specially if you use nuclear extract instead of recombinant protein.
Verna Frasca
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Hi all I performed an ITC experiment with a protein and nanoparticle. The ITC profile that I am getting is a "reverse bell shaped curve". Can anyone explain me the reason? and how to interpret it? Thanks
I would be useful to see the actual data.  As the others have commented,  you need to  make sure you do appropriate control titrations to determine heat of dilution.  I am assuming you have the nanoparticles in the ITC cell and protein in the syringe.  If nanoparticle is in the ITC syringe it should be in a colloidal suspension, other wise nanoparticles will settle in the syringe during the titration and you would be injecting different nanoparticle amounts at end of the experiment. Are nanoparticles in the same buffer and pH as the protein?  
Adrian Velazquez-Campoy
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Was anyone using nano ITC low volume (TA instruments) for binding affinity studies of protein-ligand interaction? If yes, were you getting the results properly? If yes, please give me some suggestions to get a decent saturation. I am using the same instrument but, I am not getting the saturation properly. Kindly help me with the same. Thank you, Himesh
@ Himesh, many hypotheses about your specific problem can be made, many advices can be given, but if you do not show images of the problematic titrations, you may not get the right answer to your question.
Jean Rene Grezes
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It is difficult to find which fluorophores and quenchers are off-patent, i.e. in the public domain and free to use commercially without restrictions. Would anybody have a list of these (and the number of the original patent). Their use on PCR probes might also be patented, so any information about this would be interesting. In particular, I'm interested to know if FAM and HEX dyes are off-patent for PCR. I came across this site from IDT (https://eu.idtdna.com/pages/products/modifications/fluorophores/freedom-dyes), but unsure if these are dyes that are indeed in the public domain, or are... more
Please read this link as info for F.A.M  https://en.wikipedia.org/wiki/Fluorescein_amidite Useful for further synthesis. JRG
Mohammed Amir Husain
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How to determine the distribution of DNA binding enthalpy values normalized to total drug per injection from isothermal titration experiments.
Thank you for the reply :-)
Michele Hargittai
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I have measured through anisotropy the Kd of my protein binding to the fluorescently labeled DNA as 0.2 nM. The concentration of the DNA was kept constant as 0.1 nM. I will need to increase the concentration of my DNA in a footprinting assay to 20 nM. I want to titrate the protein during the footprinting to see how the protein affects DNA structure. I am looking at starting the titration low and increasing the protein concentration until the DNA is fully bound. I cannot titrate the protein too high because aggregation occurs. Do I still stick with a Kd of 0.2 nM and base my titration of... more
It has been suggested that my protein binds dsDNA in a 1:1 ratio, but other DNA structures in a 2:1 ratio. I was looking for a good starting point for my protein titration so I can see protein binding and perhaps flush out the 2:1 binding. I agree, that in terms of DNA footprinting, protein aggregation should not be an issue, as long as DNA is still bound. Thank you all for your responses. 
Chun Guo
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Hi there, I plan to perform a SELEX on a protein target which has multiple binding site(at least two). Does anyone has experience with where can I find them? Thanks for your attention
Hi Ramesh, Thanks for your suggestion.
Yadvir Singh
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Is there any transcription factor/ sequence specific DNA binding domain, which is less than 50 amino acids in length? Can Peptides act as transcription factors?
thanks for the answers! i will try to use them instead of GAL4/UAS system. More suggestions are welcome!!
Mohammad Tarek
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I wanna distinguish the nucleosome binding and non-nucloesome binding of an nuclear proein.
http://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-10-345 this tool actually would help you to some extent if  you can re-implement the Bayes network on matlab
Girishkumar Kumaran
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Will the stability of the G quadruplex change in different buffers (for example Tris or Phosphate buffer)? What effect would pH have on the stability?
Certain G-quadruplex was found to go through conformational change at low pH. Loop was the decisive factor controlling the interconversion upon pH variation. G-Quadruplex with TT central loop could be converted in a much milder condition than the one with TTA loop. https://www.ncbi.nlm.nih.gov/pubmed/23811336
Yuri F. Drygin
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DNA enzymes and binding proteins expressed in E. coli often have bound DNA or RNA that can be troublesome to remove completely for biochemical experiments and crystallizations.
Why not to try a filtration through the DEAE-paper filter at salt concentration lower 0.4 M? 
Ataf Ali Altaf
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Eb - Ef = 0.5916 log Kred / Koxd either its oxidized or reduced component is more interacting with DNA. where Eb and Ef are the formal potential of DNA bonded and free electroactive specie.
Jai Ghosh
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I have been working on a protein which have multiple sites for binding magnesium ions. These sites are present at different domains of the same protein. How would I measure the binding affinity of Mg2+ ions to these different sites without mutating their binding sites? What kind of techniques will distinguish the binding stoichiometry of these domains to Mg2+?
Gently digest the protein sample with say trypsin and then use the different peptides obtained to see th magnesium binding characteristics.
Kristian M Hargadon
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I have been trying to study DNA binding of a yeast transcription factor under different growth conditions using ChIP. Positive control shows enrichment under a specific growth condition and that is expected. However, even negative control loci (incl. ones that do not have response element) are also getting enriched when cells are grown under same condition. The response element sequence is known and mutating this sequence results in lose of transcriptional regulation. Fixation time, Antibodies and temperature of fixation were changed to see if it would reduce non-specific enrichment.... more
Hi Stevin, I just came across this thread and am wondering whether you ever resolved this issue.  I am experiencing the same difficulties in my ChIP studies for the FOXC2 transcription factor in mouse melanoma cell lines.  Any insights you might have would be greatly appreciated.  Thank you!
Omar Gonzalez-Ortega
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ProLinker B binds to gold by thiol-gold interactions. Would the thiol-silver interaction allow a effective high-density protein immobilization as shown in gold? http://proteogen.koreasme.com/contents/products/product1.html
If the question is: would thiol groups bind to silver nanoparticles similar to gold nanoparticles. The answer is yes. Would the resulting system (silver-based) comparable to the gold system? That not only depends on the dative interaction between sulfur and gold or silver but on the resulting system, which must render the nanoparticles stability. 
Ziguo Zhang
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Dear Friend, I am after a tethering system for protein and DNA, such as laco/laci tethering system. Recently dCRISPR-Cas9-gRNA complex seems can target DNA and bind the DNA tightly, and TALEN designed zinc finger protein may also do similar thing. I wonder any of you know other system for DNA protein affinity binding? Thank you. 
Ágnes Mosolygó-Lukács
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I will perform immuofluorescent labelling in Arabidopsis. For DNA stainig I can use SYBR Gold (there is no UV laser in the microscope). Does anyone have experience with SYBR Gold in fixed Arabidopsis samples? Can anyone suggest a detiled protocol for fluorescent DNA-binding using with SYBR Gold? In human nuclei samples this staining methods has already worked in our hand.  
Peter Kapusta
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In drug - DNA binding studies ......... (Ea-Ef)/(Eb-Ef) = {B - (B2 - K2 C [DNA]/s)1/2 } / 2KC where Ea is the extinction coefficient observed for the absorption band at a given DNA concentration, Ef is the extinction coefficient of the complexes in its free form and Eb of the complex in the fully bound form. K is the binding constant C is the concentration of drug s is binding site size and B = 1 + KC + K [DNA] / 2s
Peter Kapusta
That does not change anything when K and s are fitted parameters.
Saravanabhavan Munusamy
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Hello, Please anyone explain, I am synthesis anticancer drug, but why i am analysis DNA binding/cleavage, antioxidant studies for that compound, What is the relation between DNA binding/cleavage, antioxidant and anticancer studies.
Please any one answer my question.
Jesse Jones
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I've been given pBR322 to study the DNA binding property of a synthetic peptide. How do I determine the concentration of plasmid for my absorption spectroscopy assay?  Thank you for pondering on my question. 
We use a nanophotometer in our lab (it's an Implen), which makes the process absurdly quick and easy. There are other methods, though. If you have DNA quantitation standards, you should be able to get a rough estimate from gel electrophoresis. Adam attached a really great link above that should go over all of this, though. Good luck!
David Sung Kim
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Hi, I am currently running a DNA-protein binding assay using the nitrocellulose technique. I use radioactively labeled DNA aptamers that were made using a PNK reaction (excludes the kinase method because synthetic open-end DNA). I then follow this PNK reaction up with ethanol precipitation to purify the DNA. After purification, the labeled aptamer is mixed in adequate volume of water, which is frozen for later use. For assay itself, I run an assay that binds Nogo protein in Sodium Acetate solution with the labeled DNA aptamer. Previously, binding has been present during process of... more
Asif Nakhuda
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Hello. Does anyone know of any transcription factors that have a propensity to bind to genes that are coded on the negative/minus strand of DNA? If yes, can you please provide examples and possible reasons? Thanks!
No because you are assuming there are exclusive DNA motifs on the minus strand and I don't think that has been shown to be the case. 
T. A. Beerman
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I want to do a sorting experiment by staining my live mouse ES cells with Hoechst 34580, sort them in different cell cycle phases and fix and stain each sorted cell cycle phase population with another DNA-binding dye to check if their DNA-profiles match to the gates we sorted on. My question is if I can use a dye like PI, which binds the DNA in an intercalating way, after I stained with Hoechst which binds the minor groove.Are the dyes going to interfere? Does anyone have experience with this?
I would think many intercalation agents would bind readily in the presence of Hoechst compounds.  For a reference see J Phys Chem B. 2007 May 17;111(19):5047-52. Epub 2007 Apr 25. Simultaneous binding of minor groove binder and intercalator to dodecamer DNA: importance of relative orientation of donor and acceptor in FRET.  This study examined Hoechst binding in the presence of ethidium bromide.
Shang Su
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I plan to do double cross-link for Chipping a non-DNA binding protein, and I wondered why people generally take off the first cross-linker and wash extensiveley before adding formaldehyde. Has anybody tried to just add formaldehyde for 10 min in the first cross-linking buffer?
Hi Germain Rousselet, I had done such experiment previously. I first crosslink adherent cells with 1.5mM EGS in PBS for 20 min and then add 37% formaldehyde to final concentration of 2% for annother 15 min. Then I preceded with beta-catenin ChIP. It did enhance the ChIP efficiency. Maybe the people who remove the EGS protein-protein linker in consideration of over-crosslink and you may go check the concentration and incubation time they used. Hope it helps!
Adam B Shapiro
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preparation of buffer in dmso and dna solution in dmso
Can you explain why you want to do this? I've never heard of anyone dissolving DNA in DMSO. Considering how highly charged DNA is, I wouldn't be surprised if it were insoluble in DMSO. Also, it doesn't really make sense to talk about buffers in DMSO, since it is an aprotic solvent.
Rahul Anand
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Phosphate group possess the negative charge in the DNA helix structure. is it possible to join the DNA strands laterally by removing an Oxygen. 
Thanks anyways Sir.. It was nice to interact with you.:)
Valentina Nardone
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Hi, I am trying to fix up a protocol for the analysis of protein-DNA binding with micro scale thermophoresis. My system is well-known and I can evaluate the binding with other techniques,  such as EMSA, but I would like to do also a quantification by MST. However, I cannot see the binding, even changing buffers, conditions and so on. Some experience that could help? Thank you!
Thanks for your answer! I tried with all the capillaries: standard, premium and hydrophobic, and the profiles are very similar. My proteins are very sticky, but I label the DNA and the initial scans are always very good. How was your profile with standard capillaries? How did you notice the problem of stickiness? Thanks!
Sundarrajan Balachandar
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I read a lot of articles but they do not explain it very well. I'm having trouble in finding apparent and binding constant value.
First of all, you have to focus whether it is quenching or enhancing.. then only you can able to adopt Stern-volmer plot or Benesi–Hildebrand plot..
Houda Kawas
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How to prepare Tris Hcl buffer for dna binding studies
Dear Thulasiram Bathini Pls. find the attached files, several  lab protocol and Recipes that you could select, good luck on your...more
Surya Bhushan Kumar
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I am trying to purify my protein using size exclusion chromatography (primarily by using Superose 12 10/300 column, but have also tried Superdex 200) after Histag affinity purification. The protein somehow seems to be lost during size exclusion chromatography. I either get no protein at all in the fractions, or get very little yield. I was wondering if my protein (an endonuclease) could be interacting with the matrix. Is that a possibility? How do I check for the interaction? The buffer used for SEC has 500mM NaCl, 20mM Tris and 5 % glycerol and a pH of 8.  Could there be any other reasons... more
Hi Sravya, you are using SEC /Sephacryl 200 resin  and IMAC resin. first you have to check 0.5M salt is required for SEC or not May be with 0.5 M salt enhance aggregate your protein. before SEC may be using IEX column to remove protein of interest with 0-.5M Nacl if iam correct. For that you can diafilter by TFF or dialysis bag (depend on protein Vol.) Find out salting in/out concentration of protein ? your problem is aggregation of protein on column or before column. than give appropriate buffer to soluble or monomer protein  long time. use Arginine (1-5%), Sorbitol (up to 5%), Tween...more
Steingrimur Stefansson
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I have a protein that binds DNA bubbles and that  I think is a dimer on DNA. I do not  know if one or two subunits bind to DNA. I would like to cross-link subunits adjacent to DNA (bound) to see if both subunits are bound to the same DNA bubble or only one. 
Hi John, I would recommend the book "Bioconjugate Techniques" by Craig T. Hermanson. It has a chapter on nucleic acid modification and conjugation with proteins, including biotinylation. 
Nana S Fa
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Hello everyone, I am working on hCB1 in CHO cells and I got low response of HU210  I homogenised the cells by using buffer (50mM, 2mM EDTA and 5mM MgCl2) then wash three times with high centrifuge at 30,000 at 10min. In terms of binding assay, I used the same buffer at 25C at pH 7 for 90 min (I tried 30C and 37C but there is no response). In fact, this condition works well with rat brain but not with CHO-hCB1. Can anyone give me an advice in binding assay generally?  Regards,
Can I increase the concentration of G418 ? Regards, Nahed
Manal Tawfik Hussein Mohamed
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what is the best fluorescent DNA-binding dye 
Fluoromount-G, containing 4’,6’-diamidino-2-phenylindole dihydrochloride (DAPI) for nuclear staining is good for nuclear staining
Trivikram Reddy Gundala
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I would like to use UV-Vis Spectrophotometer
Thank you so much for your response and very relevant to find the solution for my problem.  
Sugata Roychowdhury
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We are interested at looking for a protein complex that may bind DNA. The protein of interest doesn't appear to directly bind DNA (doesn't have a predicted DNA binding sequence) but localizes to the nucleus and apprears to be bound to DNA by 3D-SIM microscopy. How would we go about testing if this protein bound to DNA as part of a multi-protein complex? Would ChiP-seq be able to show that or is some other method optimal to detect a protein complex bound to DNA?
HI John, If you know the components of your protein complex and DNA sequence, I would suggest you can do gel-shift assay (also known as EMSA), better if you do DNase I Footprinting with DNA being chemically bound to fluorophore molecules. Footprinting followed by DNA sequencing will precisely tell you what DNA sequence it binds to. I am assuming you have some idea of what DNA it binds to based on the type of the protein. Now to get a quantitative measure as to how strongly the protein binds and in which order, you can try ITC or Fluorescence Anisotropy where you add protein in different...more
Tsai-Tsen Liao
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Hi guys, thanks for checking this question out. I was wondering if anyone can help me with some EMSA related questions. I have attached a picture of my recent result. 1. I've been optimizing the EMSA assay for a few weeks, what I was trying to see with this assay is the change in DNA binding activity of P53 after certain drug treatments, but so far I could not detect any difference with or without drug treatments.  For optimization purpose, I started with Nutlin-3 treatment which is known to stablize P53 and up-regulate pro-apoptotic signals such as Puma. I used nuclear extracts... more
hi Maoyu: I'm woundering did you fix the problem? since i have the similar situation, adding the antibody seems to enhance the protein/oligo binding. In my protocol,i incubated the reaction mixture with Probe first (for 10min), then i added the antibody incubation for more 20 min. Also, the density of the band will increase with the the concentration of the antibody.  Thank you so much for read my question, any suggestions will be appreciated!!
Michael Cukan
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The Wikipedia page on zinc fingers states that such domains exist; salt bridges reportedly take the place of zinc. However, there is no citation! The only example of a natural zinc-stabilized DNA binding domain that can dispense with zinc binding that I have found is Ros from Agrobacterium tumefaciens (see papers from the Pedone laboratory). This domain is stabilized by a hydrogen bond network and not "salt bridges", however, so I assume Wikipedia was thinking of some other example. I am not only interested in ββα Cys2His2 classical zinc fingers - gag knuckles, treble clefs, et cetera -... more
Sanjoy Kumar Sheet
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In 0.2 uM the absorbance is less than 0.001. 
Steingrimur Stefansson
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I am measuring the binding of thrombin binding aptamer (TBA) to thrombin using fluorescence anisotropy. TBA is labelled with a fluorophore (5'-FAM) and was initially kept at a constant concentration of 30 nM while titrating in the protein at concentrations from 1 nM to 1 uM. When fit with the quadratic binding expression (so accounting for ligand depletion), I got a Kd <10 nM. However, I then repeated these experiments with 500 and 50 pM TBA, trying to get the [TBA] below the Kd so mathematical manipulation became easier, however this increased the Kd to 27 and 127 nM respectively. (Note... more
Just to echo Adam's concerns, can you prepare trypsin (or chymotrypsin) in the same way you prepare thrombin and use that as background? 
Ning Dai
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Hello everyone, I want to know the principles of Atlas ClearSight DNA Stain binding to DNA, and the difference between Atlas ClearSight DNA Stain with SYBR Green I. Thank you.
Edward Michelini
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They bind peptides and ATP, but not DNA. What's the rationale for the naming scheme?
Originally it was noticed that mutations in these genes (dnak and dnaj) negatively impacted DNA replication and phage propagation. It was only later shown that their gene products (aka proteins) were important in the cell's heat shock response. A more useful nomenclature is the Heat shock protein (Hsp) followed by their molecular weight in kDa, or Hsp70 and Hsp40 respectively. See: https://www.wikigenes.org/e/gene/e/913406.html John: LOL
Lynda Guzik
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Hi All, I'm looking for an antibody directed against mouse nuclei (not cell type-specific), preferably  directed to component(s) of nuclear envelope, and being a monoclonal. Antibody is intended to be an alternative to DAPI staining (DNA-binding dyes need to be avoided) and will be used in FACS of isolated nuclei. Any suggestions will be appreciated. Cheers, Lech
Lech - Try the anti-lamin B1 antibodies from AbCam (http://www.abcam.com/lamin-b1-antibody-nuclear-envelope-marker-ab16048.html).  I haven't used them yet, but have a researcher who is considering use in ploidy/multi-nucleated cell experiments by flow.
Shilpi - Gupta
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hi, could anyone tell me is there any difference in sequence of five interacting partners, as per my knowledge they have common decameric binding site in target gene, what there must be some difference in sequence of homo and heterodimer
Behnaz Ghaemi
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 I can not seem to find any papers concerning this issue. What functional group can be used on the DNA to bind to ZnO NPs? what type of oligotargeter can be used to target ZnO NPs. for instance/ thiol based or amino based? reference needed please
Dear Nasr Its depend on your NPs Size and Zeta potential, usually ZnO NPs have large positive charge, so you can easily conjugate them with negatively charge molecules. But, as Sebastian said, Chemistry results are completely depend on your precursor.
David F. Barker
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Additionally, I would like to know  how the emulsion melts and how the beads are placed on a flat and solid surface? Thank in you very much in advance!  Best regards,
In SOLiD sequencing, the beads used for emulsion PCR are covalently coupled with many copies of  a primer sequence.  The DNA fragments to be sequenced are first ligated to an oligo that is complementary to the bead primer.  When the ligated fragments and beads are mixed, the fragments bind via the complementary sequences and the fragment can be replicated from the bead-bound primer.  In the emulsion PCR process, the goal is for one bead and one fragment to occur together in one aqueous "bubble' and for many copies of the fragment to be amplified while attached to the bead.  The emulsion is...more
Joseph Utermohlen
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I am currently doing DNA dot blots using nitrocellulose, however, i need to increase DNA binding thus thought of using PVDF membrane. What method of activation should I be using on the PVDF. I find that DNA resuspended in Tris/NaOH doesn't get absorb into the PVDF membrane.
See this info published on Pall website it is everything you want to know about nucleic acids and PVDF blotting.  All biomolecules stick to these membranes via hydrophobic interaction.   http://www.pall.com/main/oem-materials-and-devices/literature-library-details.page?id=27294
Jürgen Schulte
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I am conducting 1H NMR experiments on a 700 MHz machine to analyze DNA-chemical binding. The chemical I am using is an intense blue color and I have used a sub-saturation stoichiometry for the concentration of chemical. When I performed the NMR I got the expected chemical shifts but the intensity of the peak was greatly reduced (0.005 for DNA only to 0.0005 upon chemical addition). Does the color of the chemical have any effect on the peaks, and is it as significant as the peak shift? If so, what could be the corrective measure?
The strong blue color makes me wonder whether your reagent might be paramagnetic, i.e. a radical, or a Cobalt or Copper complex?
Hd Hd
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Specifically used with genomic DNA and to help prevent non-specific binding
Sven Buelow
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Are there any programs out there that can identify which specific DNA motif a transcription factor binds to, given the entire promoter sequence and TF sequence/structure. I've got the promoter sequence which I know the transcription factor binds to, and I have the  protein sequence and putative structure of the transcription factor, so have a good idea what the DNA-binding domain looks like. I'd like to be able to show specifically where in the promoter the it binds, does the software exist to be able to predict this? As a side note, does binding site of the transcription factor have any... more
As Martin correctly said, the programs only give you a first indication of what might be the binding sequence. You always need the experiment to find out. Also, some transcription factors bind far away from the  promoter boxes! Do gel band shift assays etc. A very exact, but old method is DNase I footprinting.
Daniel M Cohen
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I have a protein whose function is to reduce transcription factors. To check whether my protein is active I have to do DNA-EMSA. I am using my purified protein to reduce TF from nuclear extract from Hela cells. To do so, I am using fluorescent labelled 23 bp oligonucleotides which were annealed. EMSA was performed by binding the probe with a nuclear extract (5ug)  and running samples in 6% gel using 0.5X TBE buffer (44 mM Tris, 60 mM boric acid, 1 mM EDTA).My reaction contained 4 μl of 5X binding buffer(50%glycerol, 75mM KCl, 25mM MgCl2, 50mM Tris pH 8),5 μg Nuclear extract and my... more
@Brian- misread the thread. Thanks for pointing this out my mistake. A promiscuous motif such as the AP-1 sequence can be recognized by a wide variety of bZIP protein heterodimers, especially in the context of a nuclear extract. As such, you can fairly expect a heterogeneous population of bound probe states that would fail to produce a single discrete band. More stringent binding conditions might help (100mM KCl, 10mM MgCl2 final concentration), but I think it will be challenging to see discrete bands even under the best conditions in this experimental design.
Micah D Langford
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Hello, I am trying to find a good protocol for using these tubes. Does anyone know of any? I am targeting a DNA binding protein. I am new to this side of the sciences, so any help would be appreciated.  Thanks!
Thank you!
Rezvan Mohammadi Nezhad
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 I have a DNA-binding protein with N-terminal 10-His tag and I need to purify it while it is attached to DNA. I need high concentration of pure protein. But I can't decide between Ni-NTA column and resin. Which one is better in terms of purity and quantity? Also, does anyone have suggestions on which brand is both reliable and cost-effective?If anyone could help it would be most appreciated! Thanks!!
Hello everybody. So many thanks for your replies. In my previous research, I used HisTrap column from GE and it worked great for protein with N/C terminal His-tag but for internal His-tag, the purity was not good. Then I used Ni-NTA resin under denaturing condition. The purity increased substantially (silver staining) but the obtained concentration was low!      
Hassan Hakimi
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I am looking for the proteins  that have DNA binding motif in their structure in Babesia spp. I was wondering whether there is a free software to look for that.
Thank you Esteban.