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Hi, does anyone have a good protocol for Gata-3 staining in particular. I have 0 staining, with my protocol (BD FoxP3 staining buffer) while Tbet worked well for example?
thank you in advance
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HI...
Gata-3 is a transcription factor that plays a critical role in the development and function of immune cells, including T cells, B cells, and dendritic cells. It can be challenging to achieve consistent staining of Gata-3 in tissues or cells using immunohistochemistry or flow cytometry, as the protein can be highly heterogeneously expressed and can be difficult to detect using standard staining protocols.
If you are having difficulty staining Gata-3 in your samples using the BD FoxP3 staining buffer, there are a few things you can try to improve the staining:
Optimize the fixation and permeabilization conditions: Gata-3 is a nuclear protein, and it is important to preserve the integrity of the nuclear envelope during fixation and permeabilization to ensure that the protein is accessible to the primary antibody. Consider using a fixative that preserves the nuclear envelope (such as formaldehyde or methanol-free formaldehyde) and a permeabilization solution that is mild and does not contain detergents (such as 0.1% Triton X-100).
Optimize the primary antibody: Make sure that the primary antibody you are using is specific for Gata-3 and has been validated for use in your specific assay. Consider using a different primary antibody or testing multiple primary antibodies to find one that works well for your samples.
Optimize the secondary antibody: Make sure that the secondary antibody you are using is compatible with the primary antibody and the detection system you are using. Consider using a different secondary antibody or testing multiple secondary antibodies to find one that works well for your samples.
Optimize the staining protocol: Experiment with different incubation times and temperatures, as well as different concentrations of primary and secondary antibodies, to find the optimal staining protocol for your specific samples and assay.
I hope this information is helpful! Regards
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A transcription factor, by definition, is a: molecule that controls the activity of a gene by determining whether the gene’s DNA (deoxyribonucleic acid) is transcribed into RNA (ribonucleic acid).
So to go by definition, β-catenin should count as one, since it (through TCF/LEF) controls the expression of Wnt target genes. However, I've rarely ever see it mentioned as such. Most studies I've come across call it a coactivator of transcription factors, so I'm not sure whether my terminology is acceptable. I mean, can I call it a transcription factor in my thesis/publication?
(P.S. here are 2 examples I found where they do deem it as one:
  1. β-Catenin, a Transcription Factor Activated by Canonical Wnt Signaling, Is Expressed in Sensory Neurons of Calves Latently Infected with Bovine Herpesvirus 1
  2. Natural Products and Cancer Signaling: Isoprenoids, Polyphenols and Flavonoids <- where they mention and I quote, "β-Catenin is a crucial transcriptional factor in Wingless–Int (Wnt) signaling, and plays important role in stem cell renewal and organ regeneration")
Thanks!!
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I am working on a few transcription factors and I need to check multiple genes that they may be regulating in my fungal system. So can anyone suggest some easy to use online tool where I can input my genome sequence and the protein sequence of the TF to check for different sites that the TF may be binding to?
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Ncbi gene search interaction
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I would like to ask, I was recently doing SPR to test the binding of transcription factors to DNA. During the manual run, I found that the RU of 500nM was 110, but when I did the affnity test, I found that 500nM could not be repeated, and then I followed up The manual run can't repeat the 500nM data. I suspect that when I tested it for the first time, my DNA was placed on ice at a lower temperature. Do you think this is possible?
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yes, dsDNA and ssDNA must have different conformations of course, due to the loss of interactions between strands and freedom given to the ssDNA, that should be more linear.
just get a point on the role of your protein and see for which target its affinity is higher, ds or ss?
all the best
fred
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I have a list of transcription factors and i'm aiming to find the genes that are regulated by these TFs
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Artyom Bannikov thank you so much
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Hello!
I am just wondering how Dr. Shinya Yamanaka, in the IPSC mouse cell paper, made it possible to insert 24 transcription factors (through lentiviral vector) all into a single cell culture without killing them? Wouldn't that viral load be cytotoxic?
Thank you!
Settanan Plangsiri
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Thank you for the answer!! Robert Adolf Brinzer
Here is the link to the paper:
Are non-replicating vector virus not toxic to cell? I thought that the process of fusing viral particle into the cell and using reverse transcriptase itself could already cause toxicity even without replicating stress from the virus. However, I am not sure at all. Thank you again!!
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I'm looking for a bacterial promoter/transcription factor which can sense Acetyl-CoA.
Thank you in advance.
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Cells excrete acetate via the phosphotransacetylase-acetate kinase pathway. As they begin the transition to stationary phase, they instead resorb acetate, activate it to acetyl coenzyme A (acetyl-CoA) by means of the enzyme acetyl-CoA synthetase (Acs) and utilize it to generate energy and biosynthetic components via the tricarboxylic acid cycle and the glyoxylate shunt, respectively.
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We applied the docking test on the transcription factor, and the results showed good affinity for phenolic compounds that form hydrogen bonds with some amino acids of this factor.
How can I tell if this affinity has activated/deactivated the nuclear factor?
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It is very difficult to conclude the activation/inhibition from your docking score. You need to explore in depth in vitro or in vivo experimental validation to conclude your findings.
Best wishes,
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Hi,
I did analyzed a receptor gene for transcription factor binding sites and I found some transcription factors now I wanted to do further analysis. I would like to take suggestion regarding what I can do with this data towards a meaningful publication? also I would appreciate the help towards transcription factor regulatory network with these transcription factors. If anybody willing to help me I can plan meetings further to discuss in more details.
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Hello,
Thinking of a meaningful publication, that depends a lot on what field or organism you are working. Briefly, I would think of studying those genes and understanding their precise known function. After that, I would think of every possible relation between their biological functions and your targeted gene. You could possibly expand that analysis for searching other genes putatively regulated by those transcription factors. This depending on your biological question, quality of genome annotation, and knowledge of consensus sequences for those TF. But anyway, you would want to proceed twards describing presumable compensatory mechanisms involving such concerted regulation of TF in response to their signaling pathways and biological responses in gene expression (including your target). Validating those results by RT-qPCR would be important. Looking for transcriptome libraries that might involve modulation in those genes, even for other species, might be of some help. You can check for a possible network using STRING. You could also try using KEGG pathways for visually plotting your data, or construct your own regulatory model.
Indeed, I believe you may have to increment your analysis in some ways in order to bring more consistency to your data. I am also working with TF right now, and I would say for you to think mostly about biological processes that are regulated in response to stimuli through distinct but interconnected pathways. Know your Biology first, that's the thing. Understand your model organism, and understand the processes you're dealing with.
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If a Transcription Factor is discovered to bind to a few genes, is it possible that those genes have a relation between them? Is there a possibility that just because there is TF binding to 2 genes, can we say that those genes may regulate each other? If it is possible to assume that they can regulate each other, how can we find out those 2 genes have a realtion between them?
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1. Of course it's possible, these two genes may involved in a same or related pathways and regulated by this TF, which may regulated a biological process. But it's only a possibility and need more stronger evidences (e.g. co-expression, functional, or pathway, even the functional experiments).
2. No, there is no direct evidence that these genes could regulate each other only because of binding with a same TF, at least in my opinion.
3. If assuming that they can regulate each other, maybe you can check their co-expression, or directly knock down one genes and measure another gene expression/protein level. I'm not expert in the experiments, so no more advice could be given on this subject.
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During my current research, I need to find Transcription Factors of a gene. I have the promoters of it listed but can not find a way to research about what TF bind to that promoter of that gene. How can I do that?
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Hi Emirhan, I truly recommend the strategy proposed by Mar Muniz Moreno. If you have some coding experience, the biomaRt package in R (https://bioconductor.org/packages/release/bioc/html/biomaRt.html) allows to download the promoter sequences as a FASTA file (exportFASTA: https://rdrr.io/bioc/biomaRt/man/exportFASTA.html) which you can directly input in MEME, either the command line or web based version, as previously suggested. To add to Mar reply, once you have the MEME results, you can check your results and Transcription factors binding sites through Factorbook, a transcription factor (TF)-centric web-based repository of integrative analysis associated with ENCODE ChIP-seq data (https://www.factorbook.org).
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Dear all,
I have been facing some difficulties when trying to characterize T cell subpopulations in FFPE lung tissues using immunofluorescence. I have sucessfuly stained for T-bet and CD3, but I am not being able to stain for GATA-3 and RORgt. I have tried all protocols that I could come up with, including the ones using signal amplification with streptavidin- and tyramide-conjugated fluorescent dyes. I have tried different dilutions of primary mono and polyclonal antibodies from different vendors; same thing with secondary antibodies. None of them has worked for these two transcription factors, even after overnight incubation. I have also tried to use Triton-X-100 in my buffer when diluting the primary and secondary antibodies, and different wash buffers (PBS and TRIS added with Triton X-100 or Tween 20). So far, I have only performed antigen retrieval using the Dako PT Link HIER system (high and low pH buffers). What intrigues me the most is that all transcription factors are easily detected when using bright field immunohistochemistry staining (DAB chromogen), meaning that the antibodies do penetrate the tissue and bind to the transcription factors. So, the problem seems to be on fluorescence, but I cannot figure it out.
Does anyone have experience with staining FFPE tissues for these transcription factors? I would really appreciate any tip that can get this assay to work.
Thank you.
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Update:
I have tried a TSA protocol again using a GATA-3 primary antibody by Abcam (ab199428), a horse anti-rabbit IgG polymer (part of the ImmPress kit by Vector Labs) and a Cy5 dye by Biotium (equivalent to CF647, cat. no. 96022). Apparently it worked well. Since GATA-3 is highly expressed in my test tissue sample, a dye dimmer than Cy3/AF555 or Cy2/AF488 could be a better option.
I am now try to get RORgt to work and will hopefully come back with new updates soon.
Cheers.
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I have a list of significant SNPs and would like to perform a Motif Discovery and Transcription Factor analysis.
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Weblink for ReMap:https://remap.univ-amu.fr/
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I want to do Chip assay for detection of three different transcription factors forming a complex and bind to DNA at the same locus.
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You can prepare your sonicated, cross-linked chromatin and then divide into three aliquots (four including the IgG control). Incubate each with antibody of choice, pull down (lots of kits for this, Active Motif has a straightforward one) and then qPCR the locus of interest. If all three proteins are bound there, all three antibody pulldowns should give a qPCR result much higher than background (IgG). Separate assays would likely have to be used to validate that those proteins are part of the same complex. I'm not aware of the assay to simultaneously confirm the presence of proteins in a complex and pull down the entire complex to assess bound DNA loci. If it is being done it seems that it would be quite complex/finicky and dependent on each unique protein complex.
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Hello all, I have been working on in-silico analysis of plant TFs. I found that there is a numbering after each TF like OsWRKY1, TaDREB15, etc. I am not able to find out how to assign these numbering (1 and 15)?
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Dear Pankaj,
Most of the time, names given to TFs are based either on their chromosomal location or on the previously reported dataset.
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i am using the protocol written by Britta Blumenthal (2011) on a bacterial transcription factor. My lanes are smearing and getting stuck at the stacking/separating interface. How do I improve my resolution? I run for 30min at 150V (15mA) then 250V for 2-4 hrs (2-4mA). Gel is 4-15% gradient with a 3.2% stacking.
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Hey, I was also facing the smear problem initially when I started running the BN page for membrane protein samples.
I solved it by adding 500mM 6 aminocaproic acid while casting the 4-15% gels. Also, I added 500mM 6 aminocaproic acid to the cathode buffer(both with and without dye).
This solved the smear problem as the protein got solubilized better in the gel.
Moreover, run the gel at 100V initially until the samples leave the wells and enter into resolving and then increase the volts to 200V and run for 2-4hrs.
Hope it helps
You can refer to BN-PAGE nature protocol for further insights; doi:10.1038/nprot.2006.62
Hope it helps.
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I made several combinations of mutations in a 747 amino acid-protein based on prediction tool as well as some additional mutations in non-predicted lysines including one affecting all 42 lysine of the protein. I did not observe a phenotypic change specific to K-R mutation, but I think the effect of fluorescent tag used here is more dominant in the protein stability. I am wondering if anyone has also observed the effect of changes in amino acid side chain geometry and electrostatic interactions in such R variants.
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Annemarie Honegger Thank you for your suggestion! I appreciate it. Hopefully David Vukovic will be able to provide me specific answer.
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Is there a way to find repressor/inhibition factor for a gene? Is there a website that list them? I found it to be much harder to find compare to transcription factors...
Thank you!!
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Well, on the browser page in hg19, there are a lot of track options, find the TFBS and choose the way to see them, refresh and you'll see it in the browser.
all the best
fred
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I've got a list of genes that I would like to find transcription factor for. But when I looked the gene up on Genecard, there are so many to choose from (on some, the list goes up to 100s of TFs!). How do I select the transcription factor that would be effective??
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Settanan Plangsiri On the same website, they provide a TFBS prediction tool. Just paste the promoter sequences of HBA2 and the tool will give you a list of TFs and corresponding binding sites.
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I am setting up experiments to look at redox interactions between Ref-1 protein and transcription factors upon different treatment conditions. I am looking for western blot protocol for native gel condition.
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Hello Mahmut Mijit,
Transfer of protein from native PAGE is uncommon. Even if you go ahead with native PAGE, your protein in the native gel do not have SDS. Under this condition, protein transfer to the membrane is dependent on the SDS present in the buffer used for soaking purpose just prior to transfer.
Hope these publications could solve your issue. See the following link:
Native Electrophoresis and Western Blot Analysis (NEWeB): Methods and Applications. . 2015;1312:343-53.Methods Mol Biol
doi: 10.1007/978-1-4939-2694-7_35.
Go though the detailed protocol given in the published paper (dx.doi.org/10.17504/protocols.io.bu67nzhn) by Christin Köbler et al entitled "Native gel electrophoresis and Western Blot transfer of Kai-protein complexes."
Best
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I want to perform Luciferase assay to check the strength of the selected promoter and binding of the transcription factor.
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I agree with Dr. John Hardy Lockhart.
As a small comment, RNA contamination is often a problem in downstream studies. See the same web page on its removal.
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Hi all,
I am currently looking into how the presence of an oncogene influences cytokines responsiveness (primes cells to be more responsive to cytokines) within RNA-seq data. What elements and tools should I be looking into?
Would upregulation transcription factors be something that would explain such a phenomenon? If so could someone direct me to a resource/literature that would possibly explain the relationship between transcription factors and cytokines responsiveness?
Thank you!
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Look at the properties of the STAT family and its canonical and non-canonical pathways.
good luck
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There are numerous articles on this topic already by google. Just to wonder are there some tools that are frequently used by biologists and can be related to experimental measurement?
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you can go to the UCSC database to see your favorite genes or positions, in front of the selected data in the optional lists, and also send your own data to the genome browser (http://genome.ucsc.edu).
all the best
fred
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Need to establish the assay development of phosphorylation of a transcription factor that contributes to prostate cancer. I want to develop the assay manually rather than using any Kit.
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Very first, try to find out what is already deposited in databases!
A surprising amount of data has found access in publicly available databases, (NCBI.org) often containing information that has never been formally published from mass spec experiments. Look for the latest protein GenBank (.gb) entry of your sequence, it will contain the known information in the annotation section there you may get a first impression on whats known, which can save a lot of unnecessary work . Then you can go ahead and mutagenize the site and investigate your biology. Also Mass Spec is a fast way to map a site, it may also reveal a lot of additional information on other PTMs of your protein of interest:
  • DOI: 10.1016/S0076-6879(02)51853-X
Good luck! Thomas :-)
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Specially, I am looking for a database or software which give me the name of transcription factors and mRNAs which are being regulated by them.
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Thank you so much for your good recomendations.
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Hello everyone,
I am looking for a nuclear marker, like a transcription factor, expressed in human enteric neurons.
I would like to characterize my human iPSC-derived culture with immunofluorescence stainings. The problem is that my neural progenitors give rise to enteric neurons and enteric glia (GFAP+), so I cannot use Sox10 throughout the whole maturation. So far I haven't found anything this specific.
Does any of you have any suggestions about a good antibody, or a marker that would be useful in this case?
Thanks a lot in advance.
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Dear Alexandr,
Thank you for the answer. Unfortunately, I already know these works really well!
I can't use neural crest markers in IF as most of them are either cytoplasmic or downregulated before the enteric neurons become terminally differentiated.
Sox10 works quite well up to a certain point, but it's absent in mature neurons: its expression is maintained in enteric glia only.
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I want to use this system to identify transcription factors regulating my target promoters.
I already constructed my baits and tested the background expression.
Following the transformation protocol (small scale) provided with the Yeastmaker™ Yeast Transformation System 2, I first tried to construct my bait strains a few times, but without success. I could not get any clones.
I finally could create my bait strains, using a “quick and easy” protocol, without any problems.
I tried a test screen with the positive control (S.c. Y1HGold [p53/AbAi]), following the manufacturers protocol, but again without success. There was just one clone on a SD/ -Leu Plate (1:10 dilution), but no clones on SD/ -Leu +AbA (screening media), which mean far too little clones. :)
I would be really pleased, if anyone would have some suggestions according to a working protocol for a library screen.
Thank you in advance
Sebastian
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@Sebastian,
I had a hard time transforming my pBait-AbAi vector into Y1H gold. Can you share with me the 'quick and easy' method? Because the link you shared is not valid anymore.
Thanks in advance.
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Hello, i want to get all transcription factor gene list in Brassica rapa. But I don't know where I will get. I found gene in BRAD but I can not download.
Could I get any link from where I collect all TFs gene in Brassica rapa?
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yes, you are right. another way, simply you click on brassica rapa then download all protein sequences of transcription factors. Secondly, these protein sequences search in the original brassica rapa genome. hopefully in this way you find the original name of these genes. Stay blessed.
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I have selected two transcription factors whose activity I want to measure by luciferase reporter assay. For this do I have to prepare two constructs, a: Luciferase gene with a transcription factor promoter, b: another construct with the transcription factor gene? and have to perform cotransfection in HEK293T cells in order to measure specific transcription factor activity.
Any suggestions would be highly appreciated.
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Neha Khandelwal, I think your plan is correct. You need to clone the target promotor sequence of your transcription factors, upstream to the luciferase gene of the luciferase vector. Or else you can check whether they are commercially available at addgene (http://www.addgene.org/). If you plan to clone the promoter, several consecutive repeats of the promotor sequence might give promising results of the activated transcription factor. For example commercially available pNL3.2.NF-κB-RE reporter vector for NFkB activation, contains five consecutive CGGGAATTT and CCGGGGACTTTC NF-κB promotor sequences alternatively. Then you need to co-transfect both the transcription factor overexpressing vector (probably pcDNA3.1 (+)) and your target promoter-driven luciferase vector at the same time.
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Hello,
I had a question about the CHIP-seq analysis. Do you think that if you do a CHIP-seq of a protein, and you get peaks, this protein can be confirmed 100% as a transcription factor? Or could it possibly be a cofactor/signaling protein? In other words, it did not have direct DNA binding site, but when fixing, the cofactor/signal protein remains bound to the TF, and then when performing the analysis, you obtain peaks of a cofactor that does not actually have a DNA-binding domain. Wouldn't it be necessary to confirm it with an EMSA or some similar in vitro test? For example, in this case performing a CHIP-seq of Notch1, (https://www.pnas.org/content/111/2/705) obtaining peaks but not being a TF as such, but rather a ligand of RBPJ (Image from: https: //www.pnas.org/content/108/36/14715.)
Thank you so much! =)
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CHIP-seq is not specific for transcription factors. Two commonly used standards for CHIP-seq, H3K27Ac and RNA polymerase II, are not transcription factors and instead provide insight into chromatin status and transcriptional activity.
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I recently performed RNA sequencing on wildtype and knockout mice and have identified several differentially expressed genes (over 1,400 DEGs), and I'm interested in identifying what transcription factors are known or predicted to influence the transcription of these genes. Are there any bioinformatic tools or databases that can help me identify or predict those transcription factors? I've heard of AltAnalyst while attending a conference, but I can't seem to find it or any documentation. Any help would be appreciated!
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I prefer the hESlincRNABrower- you can use it
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I’m planning to investigate the variability o f the mixed layer depth in the Moroccan Atlantic cost using CTD data. I would like some advice about recommended techniques. I’m really looking to find out what you’d consider to be the most efficient method.
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You may be interested in Holte and Talley 2009 paper mentioned here http://mixedlayer.ucsd.edu/
The algorithm is part of the Climate Data Toolbox. It is based on both the threshold and gradient methods for T, \rho and S, and chooses the "best" one.
I just found this and plan to use it.
Cheers
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What program allows add annotations on a double-stranded DNA sequence (e.g. transcription factor binding sites) and export the prepared sequence as an image? I need such an image for my research work.
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I don't know what you mean by annotation, but you can try UniPro UGENE.
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Hi,
I´m investigating the homo- and heterodimer formation of two proteins (A & B). For further results, I would like to perform experiments to understand where the homodimeric complexes (A-A, B-B) bind to DNA in comparison to the heterodimer (A-B). I think that this question can not be answered by performing traditional ChIP-Seq analysis with anti-A and anti-B-antibodies.
Does anybody know if there is an appropriate method for answering this question?
Thank you!
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One thing you can do is DNase footprinting matched with a motif analysis depend on if they have different binding motifs or not. You can also do a sequential ChIP experiment with Anti A and then Anti B. This should give you the regions were both are bound. Then you can do a ChIP with just Anti A and/or just Anti B to find overlapping and unique sites.
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For transcription factors co-locate with enhancer DNA sequences
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Hello, for detection signal above threshold level and appropriate number of Ct value for fish fluorescence you need to have at certain amount of probes at initial stage.
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Hi all, I am trying to expressing zinc finger transcription factor in BL21(DE3) RIL cells. It is tagged with an N-terminal S-tag. I tried different concentrations of IPTG (0.1-1 mM) and different temperatures (16-37 degrees C) but I always end up with quite a strong band in the insoluble fraction. I am using LB medium. I read a few suggestions to use Zn or DNase during extraction. Does anyone have an idea what could help? I would like to avoid purification from the insoluble fraction because the structure of this transcription factor is not known. I need it for pull down assay with another protein.
Thanks a lot for any suggestions.
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You may try SoluBL21, developed by and available from Genlantis http://www.genlantis.com/solubl21-competent-e-coli.html. If needed you can transform it with the RIL plasmid for rare codons. It may not work for your protein, but it is still a good strain to keep in your collection for further recombinant protein projects.
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For example, (TF) YY1 = inflammation responsive/ metabolic responsive/cell cycle regulation, which option is correct? Please, suggest any database links, where I can easily find the type of transcription factors.
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Thanks a lot Aadil Javed sir
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Im researching the role of gene TRIM58 in enucleation process in hematophoeisis. I did already a knock-out of TRIM58 but i would like to do a cell line where i overexpress it too. What would be the best way to do so? CRISPR coupled with a transcription factor? Any other suggestions? Thanks for the help
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Please see below the workflow for theTRIM58 Stable Cell Line Generation -Host cell line: You may choose any (HEK293T or CHO-K1, or other cell types are also available according to your requirements) -Vector construction -Pool cells generation -Monoclonal cells screening and identification -Validation by qPCR -Mycoplasma detection
Have fun and Happy research!
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NFKb is a transcription factor that activates various genes. We have found the differential regulation of Pax6, as well as, NFkb in response to UV treatment under in vitro conditions. Can someone help me to design experiments that will help me to find out if NFkb is binding to the promoter of Pax 6 gene?
Thanks a lot!
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ClusPro is software that can help you in finding if they can bind via generating a binding model for them.
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For the study, we have isolated a promoter sequence that has been ligated with Luciferase vector and transfected into the HeLa cells which are being incubated in different experimental conditions. Luciferase assay will be used to predict if the gene had a direct or indirect impact on certain processes on our model organism. For that very purpose, I have been looking for the transcription factors or other motifs that can bind to our promoter sequence, but the search for the software has been in vain. It would be really helpful if anyone could suggest some resources for the prediction.
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Hello,
You can do it using ALGGEN PROMO, it is easy and straight forward just have to put your promoter sequence, indicating the species, factors, and Matrices. It will give you both predicted and validated transcription factors for your sequence.
Good Luck
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Hi everyone,
I have a list of differentially expressed genes from which I want to :
  • Identify the potential transcription factors involved in the regulation of my differentially expressed genes.
Could you please suggest some tools/R packages or platforms to perform this kind of analysis?
Thank you.
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The short answer is that this is not easy nor are there accurate ways to gauge TFs from a set of differentially expressed genes. For starters any DEGs may be directly affected by a TF or indirectly affected by something else regulated by a TF. You can search promoters for consensus sites or patterns but again this is not easy. Sometimes you have to do an experiment to get the results.
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I'm analyzing transcription factor binding motifs and some of them appear with a double colon symbol "::". For example: Ahr::Arnt
I found explanations for transposons, fusion proteins and others, but not for transcription factors.
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Hi Amaya,
The double colon in Ahr::Arnt means a dimer of Ahr and Arnt.
Hope this helps!
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I have the Chip Peak calling sequence data for a gene, and I need to find out Transcription factor binding site at the promoter region of a gene. I have selected the EMSA to find out whether to check the transcription is binding to the promoter region..Since I have many gene, is there any way to use the chip peak calling sequence to find out the Transcription factor binding site..
Thanks in Advance..
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You're welcome Jayalakshmi Thiruppathi let me know how it goes
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I was wondering whether it is possible to turn off the expression of a transcription factor that is expressed by CD8 T cells, and if yes, how?
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Yes it us possible. You can design a sgRNA and then use CRISPR can system to knock out either the promoter region or the main gene of the transcription factor employing NHEJ mechanism of cells which is error prone and induces INDELs
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What is the best approach to identify TFs in a non-model organism with limited homology to the closest model organism? Is predicting DNA-binding domains and assuming that most proteins containing DBDs are TFs a valid strategy?
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You can run your proteins with InterProScan and check the domains.
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I would like to know about all the molecules that can be expressed by/in CD8 T cells
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Hi Fa,
Your question appears simple but there are several answers depending on the species (human vs mouse) and the state of the CD8 T cell activation.
Naive CD8+ T cells express CD62L (L-selectin) integrin, IL-7Ra (CD127), S1PR1 and CCR7 chemokine receptors.
They also express the costimulatory molecule CD28 and negative regulators of T cell activation such as TGFbRI and VISTA. These are all surface markers but they also express specific transcription factors such as Foxo-1 and KLF-2
Just after activation and during priming, CD8 T cells upregulate the expression of CTLA-4 and CD69 then later PD-1, and LAG-3 as well as Fas. As the CD8 T cells are primed for cytotoxic potential, they express Granzyme B (GzmB) and Perforin.
CD44 is an excellent marker for antigen experience of mouse T cells.
However, human CD8 T cells are different since naive cells are CD45RA+ CD45RO- while effector T cells are CD45RO+ CD45RA-
However, there are many more molecules than those I just listed. For a more comprehensive knowledge of these, please refer to these resources:
2-ImmGen is also a terrific resource where you can check for the expression of specific markers
There is one minor, yet important point to consider. The classically defined CD8+ T cell subset is the αβ CD8 T cell. However, there are also αα CD8 T cells which are part of intraepithelial T cells in the intestines.These cells do not express the β TCR subunit and their role in gut homeostasis remains unclear.
I hope this reply was of any help.
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I have several different transcription factors including Laci and TetR that I have expressed and purified. I have tried several different buffer conditions including PBS, HEPES, and Tris. I have also tried different storage temperatures. All of my proteins start to denature within several days.
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The best way is to mix it with 50% glycerol and keeps it stored at -80ºC. Be careful in each step, don't allow it to completely defrost immediately after using it.
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I am working with a very unstable transcriptional factor. I use DNAse in the lysis buffer and I purify it as inclusion bodies, by using urea and 1M NaCl. After purification, I buffer exchange the protein to 50mM Hepes pH8, 1M NaCl. In this buffer, the protein is stable around 0.5mg/ml. The 260/280 ratio is 0.6.
Nowadays I am working with a N-terminal truncation of this protein. I use the same protocol, but the 260/280 ratio obtained is 1.5. As I purify it by using urea, I assume that it is not possible that my protein has DNA bound. But I cannot explain this 260/280 ratio?
Any ideas?
Thanks
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You could try to quantify both the amount of protein and the amount of DNA in the sample. A standard protein assay such as Bradford or BCA would do for the protein measurement. I don't think a small amount of DNA would interfere. For the DNA measurement, you would have to denature or destroy the protein by boiling it or treating it with protease, then purify the released DNA from and measure its concentration by standard means.
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I want to visualize EOMES, a transcription factor, in CD8 T cells. I am planning to perform an immunofluorescence test and I was wondering what kind of stain or chemicals I can use to enable the visualization of internal markers of a cell.
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Fa Ro , that does make this quite a bit more complicated.
Fluorescently-conjugated small peptides (i.e. phalloidin) can be used to visualize some intracellular targets in live cells (actin filaments in the case of phalloidin), but this does not apply to most proteins. Unfortunately the size of normal antibodies prevents their entry into live cells, but a few different approaches for labeling intracellular targets in live cells (e.g. quantum dots and nanobodies) have been published. Unfortunately, none of these systems are in widespread use.
The most common approach for live cell visualization of a specific protein is constructing a fluorescent fusion protein. This can be done with transient transfection or through generation of stable cell lines. However, there is no guarantee that the fusion proteins will behave in the same manner as the wild-type due to steric hindrance.
One group has already reported a GFP-Eomes fusion protein in mice: . They used TALENs to insert the GFP into the first exon of Eomes, but you should be able to reconstruct this in a plasmid.
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Is there any database that can provide me with information on how a particular gene is regulated (for example, names of transcription factors)
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awo! great, thanks
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Why these transcription factors have fused name from two different protein For e.g AP2 play their role in flower development and ERF is ethylene response factor, But in ABA signalling in abiotic stress. We write them together as a single transcription factor protein.
One answer could be that this is different protein but shared the conserved domain or motif of AP2 and ERF.
Second answer could be that this is the superfamily name which consist both transcription factors in it. But why we mentioned it like a single protein (see in figure )in ABA signalling.
I need some clarity.
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This figure
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When I studied the expression of Hypoxia Inducible transcription factor 1a from the whole cell lysate in western blot analysis, I got its expression. But when I isolated the proteins from cells using a nuclear lysate protocol, then there was no expression of the protein.
I had induced hypoxia using cobalt chloride at 200uM concentration for 24hrs.
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Thank you Eugen Werwein for your repsonse.
No, I did not have histone staining to confirm nuclear protien. I only had a beta actin.
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Upon overexpression of the transcription factors, the luciferase reporter activity is increasing and upon knocking down or inhibition of the selected transcription factors decreases the reporter activity. However, the endogenous level of protein of interest is contradictory to that of the reporter activity. This is happening with all the selected transcription factors.
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Transcription factors have fine-tuning and the plant tries to maintain a certain level of protein through degradation to reduce transcription factor activity. In contrast, luciferase does not possess this feedback within the cell and thus is accumulated, with a lower level of degradation. In this way, reporter protein activity is excellent in short analyses, while it loses significance in long-term analyses when there is this type of protein degradation feedback.
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Is there any evidence that explains this transcription factors behavior, i mean, perhaps structural evidence regarding the type of transactivator domain or perhaps is only a matter of DNA binding stability?
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Hi Oscar,
Zinc fingers are unique amongst transcriptional factors, which normally bind to palindromic sequences of DNA following dimerization. The ZF instead coordinates with Zn ions to literally form a finger-like projection that binds to the major groove, linking other ZFs linearly in tandem as they do so. This feature is actually being capitalized upon in the pharmaceutical industry.
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Hi,
could anyone recommend a plasmid and/or protocol for reporter gene assay in S. cerevisiae? I want to assess the effect of growth conditions on a transcription factor, so I want to clone it`s recognition sequence in front of a minimal promoter followed by reporter gene. Preferentially, I would use a luciferin or lacZ reporter.
Thank you.
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Hello Michael,
Visiting "addgene" web page at the link provided below might be of help to you.
Good luck
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I am working on transcription factors. TF are localized in the nucleus to control transcription. But many transcription factors localized to the endoplasmic reticulum or other cell organelle instead of going to the nucleus. How and why????
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There are membrane bound transcription factors that are found in the ER and associated with plasma membrane before transport to the nucleus. This article has a good description.
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Hi all,
I stained tdTomato+ cells (PBMCs and Jurkats) with eBioscience Foxp3 / Transcription Factor Staining Buffer Set for the foxp3 and I completely lost the signal of tdTomato. I tried to fix them first with 1% PFA (10min RT) and then permeabilize, which didn't work, and if I only fix them with PFA I can see the signal so I'm assuming that something on the kit is interfering with the detection of tdTomato.
I was wondering if anyone had the same problem and if yes any ideas?
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Permeabilization buffers such as TritonX100 or Tween-20 usually denature the fluorescent proteins such as TdTomato or RFP, GFP. As Urs Mörbe suggested if you want to combine TdTomato with antibody staining you need to use an anti-TdTomato antibody to restore the signal as antibodies readily bind to denature and no longer fluorescent TdTomato.
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I have a gene with a SNP in the promoter area. There are several transcription factors binding to the locus. How to find the difference in transcription level of the gene in cells with the mutation and in normal cells?
This area is rather new to me and I'm not entirely sure how to approach this task.
Thank you in advance!
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I agree with Eugen Werwein ChIP would be a good way to address this but is a bit of a complicated assay. Since you have only one region of interest you should be able to do ChIP-qPCR which is relatively cheap compared to sequencing methods. If you want to use ChIP you can either tag your TF or get an antibody against it. Then you will want to design primers to determine if you are able to immunoprecipitate the region of interest with or without the SNP.
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Hello Everyone, I have identified some genes through QTL Mapping, now i want to do analysis to find out related genes or proteins, , cis and trans element, functional variations in gene's promoter, interacting proteins and transcriptional factors.
Who can guide me about above mentioned analysis.
i will be very grateful for your kind assistance.
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Dj Jd, , Aditya Banerjee and Asif Khan Thank you so much for kind help.
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I have to control the expression of a gene ( in vitro ). For designing an optimal promoter assay, I would like to know more about Transcription factor binding region, enhancer and repressor elements on the promoter region.
Can anyone please suggest me good tools or methods to do the same.
Thank you,
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Thank you for your response. I will check out your suggestions
.
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According to information from online database [MRTFA - Myocardin-related transcription factor A - Homo sapiens (Human) - MRTFA gene & protein](https://www.uniprot.org/uniprot/Q969V6) and [MKL1 protein expression summary - The Human Protein Atlas](https://www.proteinatlas.org/ENSG00000196588-MKL1), the molecular weight of MRTF-A (MKL 1) is 931 aa, 99 kDa.
However, the western blot predicted band size of antibodies ranged from 109 kDa (Anti-Mkl1/MRTFA antibody ab49311) to 145 kDa ([MKL1 Antibody 21166-1-AP | Proteintech](https://www.ptglab.com/products/MKL1-Antibody-21166-1-AP.htm) and [MKL1/MRTF-A Antibody | Cell Signaling Technology](https://www.cellsignal.co.uk/products/primary-antibodies/mkl1-mrtf-a-antibody/14760?N=0+4294956287&Nrpp=200&No=3400&fromPage=plp).
The predicted band size ranged from 109 kDa to 145 kDa. I am wondering the reason. How do I choose the appropriate MRTF-A antibody?
Thanks a lot!
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Check the source of target protein. which species (Human, Mouse, Rat) the antibody reacts with?
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e.g., the level of the transcription factor "TP53" in Alzheimer's disease?
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hi,
you can also go to GEO profile from NCBI and search for a study on AD. typing the gene in those studies will give you some data.
fred
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Hello Everyone, 
I have bunch of RNASeq data of differential expressed gene. I wanted to find the transcription factor binding site of these gene. How can i find? Do i need to see the pathway also? Thanks  
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Yes it is possible. You might need to look for ChIP-seq intervals which are closer to your RNAseq interval.
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Actually, I know a mRNAs ID which, arises from a single gene locus transcription factor locus number and now I want to know about a tool (for plants) that would be helpful for the conversion of that locus ID into gene ID.
I also tried DAVID gene ID converter, Ensembl Biomart, Database to Database Conversions but they all are for the animal, microbes not for the plant purposes.
Thank you very much!
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maybe you will find your answer. Good luck.
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Hi, I have a question if anyone can answer. I have identified two co-factors for my transcription factor. But the mass spec data seems insignificant. Now I want to find if some other co-factors are involved in regulating my transcription factor? So, how can I do this? Should I cut my gel band at different molecular weight or should I go for ChIP-seq to determine their binding complex?
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Do you have any idea what these other co-factors might be? if not ChIP-seq would not really be possible if you don't know the protein of interest for the cofactor. At that point I would think about trying other methods.
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I came across the following claim: The general transcription factors seem unable to assemble onto a promoter that is packaged in a conventional nucleosome. In fact, such packaging may have evolved in part to ensure that leaky, or basal, transcription initiation (initiation at a promoter in the absence of gene activator protein bound upstream of it) does not occur.
What are some gene examples or how could I go about finding them?
- from Molecular Biology of the Cell. 4th edition. by Alberts https://www.ncbi.nlm.nih.gov/books/NBK26872/
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Many different cell types completely turn off genes that are not required. Especially if they are included in the heterochromatin region. You can explore this using ENCODE, Roadmap, and many other datasets. Is this what you were asking?
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Hi, I am interested to know the DNA sequence of the TF corrisponding to the DNA binding domain.
Do you know any tool developed recently that can help?
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1) If you already know the AA sequence of the DNA binding domain, you can find out the start and stop site of this domain in its entire protein sequence. For example from 101 AA- 150 AA, which is translated by the 301 nt -450 nt in the coding region of its cDNA.
or 2) You can reverse translate the AA sequence of its DNA binding domain into degenerate nucleotide sequence, and find it out from the cDNA of your interest or BLAST it against the cDNA of your interest. For example, I have a cDNA sequence with ORF: ATGACCGTTGCCAGCAAATGCgcgtgcgatgaatttggccatattaaactgACGGATCGATACGTACAGTAA, if I need to find out what the exact cDNA sequence corresponding to the AA sequence ACDEFGHIKL, I first reverse translate the AA sequence into gcntgygaygarttyggncayathaarytn (if Snap gene can't do the reverse translation, you can use online tool https://www.bioinformatics.org/sms2/rev_trans.html ), this sequence can be used to search (if Snap gene software allows you search sequence with ambiguity), blast or align against the cDNA sequence.
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I'm analysing a gene promoter sequences to predict transcription factors. I used two different tools that gave me contradictory results. I need a third option tool to help me confirm the good result.
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CiiiDER ConTra v3 TrFAST LASAGNA-Search Ahmed Afailal Tribak
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Actually, my question is: can the transcription factor bind to RNA?
Do you know a tool for finding transcription factors that bind to RNA?
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It would depend on the binding mode - frequently, Many transcription factors recognize the sequence on double-stranded DNA - in RNA, dependent on secondary structure, the local structure may be quite different.
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Hello,
I was wondering if any of you have some experience with ChIP experiment on transcription factors that would only have a few targets and if there are some specific adaptation that would be needed compared to a classical ChIP experiment.
I did some search on the literature and I could not really find anything about it. For what I understand ChIP is usually done on transcription factors having a lot of targets genes.
For my potential experiment I would like to test if a protein can bind the promoter of some genes but there would only be a few targets.
Thank you for your help !
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Joshua Beytebiere Thank you, for the suggestion !
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we are doing flow cytometric analysis of mouse T lymphocytes. We want to check the nuclear transcription factors. Currently we are running low on PMA and ionomycin. Is it possible to do the analysis without them?
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Hi Sreyasi,
Yes. You can analyse expression of transcription factors in T cells without PMA/Ionomycin treatment. This is required to enhance detection of intracellular cytokines, but not for transcription factors.
I agree with the other comments and suggestions about the need of stimulating cells, especially if starting with naive T cells.
Here is a link with protocols for intracellular staining:
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Hello all,
I am trying to overexpress a oocyte-specific transcription factor in HEK293 cells. But it never worked in protein level, I cannot detect it through western blot but I do detect it through RT-PCR. So the construct in under control of CMV or EF1a promoter neither of them worked in protein level. When I tried to overexpressed, I have another gene which was cloned into the same vector, it can be detected in protein level by western blot, which can be considered as a positive control.
Then I tried to use lentivirus to infect different cell types, including primary bovine cells, HLP-1 cells and HEK293 cells, still doesn't work the lentivirus promoter is also CMV promoter. I am wondering if anybody have any idea.
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Juste ensure that the stop codon has been removed from the insert to get the tag translated. If it's ok, try to put a N-ter tag to your insert. If you have a bacterial version, this means that the insert is fine. Did you use this very one for expression in mammals? Last, although unlikely, the protein might have a rapid turnover (treat cells with MG132 to stabilize) or cells expressing the factor may die or detach (you may reduce the period of recovery after transfection).
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Binding of cytokines and some growth factors to their receptors activates STAT3, which is a type of transcription factor. How to understand that at a certain time, is STAT3 activated in the cell?
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Interesting question. Following the discussion.
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I have a gene (Homo sapiens) and I want to find transcription factors for it. Which tools can help me. Also, If I find TF binding sites, how do I proceed after getting TFBSs. As in how do I get TFs from TFBSs? Any help is highly appreciated. Apologies for this question. I am new to this area.
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You can find the target TFs from your TFB sequence. Please go through the following link:
It has a series of RNA research tools.
All the best,
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The question might be a little bit stupid but I haven't found direct answer yet. Maybe anyone can explain it.
Why AHL molecules do not induct the activation of quorum-sensing genes inside the cells where they were synthesized? I mean, they might bound with transcription factors without leaving the cell, at least for gram-negative bacteria.
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That is because quorum-sensing is threshold-dependant: every cell produce an amount of molecules that cannot trigger it inside the same aforementioned cell. So, basically, if a lot of bacteria produce those molecules that means also that their intake will exceed the threshold value and cause the phenomenon to be activated. It is a fine and logic way for bacteria to communicate the size of the microbial population. Hope that helped
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Hi
I am studying the transactivation and the transrepression of Liver X Receptor (LXRs) in COS-7 and RAW264.7 cells. I transfect 300ng of LXR response element LXRE with 50ng of LXRs or pcDNA3.1 using X-treme gene. I treat the cells with 1 μm GW3965 for 24 hrs. I incubate the cells in medium supplemented with 0.5% delipidated serum. after 48 hrs of transfection I measure the luciferase activity. The problem is that I am getting a response in the pcDNA3.1 transfected cells similar to the LXR transfected cells. I am sure that my plasmid constructs are working as I tried them in western blot and I sent them for sequencing. I tried to re-prep my plasmid construct and I tried using different pcDNA3.1 concentration and I noticed that it affected the read out. does anyone know why the pcDNA3.1 is affecting the LXRE response? and is there any protocol that I can follow to do my luciferase assay?
Thanks
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Hi Razan, did you figure out the problem? I had the same problem with pcDNA3.1. I was thinking to change another backbone vector? Do you have any good suggestions? Thank you.
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I am trying to verify the presence of a transcription factor's consensus sequence on the promoter of my gene of interest. I have the gene sequence but I do not know of any software to check if the transcription factor's sequence is on my gene. This TF has various motifs, so I cannot tell manually. Thank you
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If you already know the motifs and want to locate them in your gene sequence, you can try FIMO in the MEME Suite: http://meme-suite.org/
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I want to study the effect of inhibition of proteosomal degradation machinery on the expression of a particular Transcription factor which we assume is degraded by ubiquitin mediated degradation on MCF-7 and A549 cells. What concentration of MG132 should I use and how long should I give the treatment. Your feedback will be valuable. Thanks!
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Hi Vishal,
concentrations around 1-25 micromolar and treatments from 2-12 hours should be OK. If you want a starting point I would use 10 micromolar for 4 hours
Hipe ir helps
Francisco
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