Transcription Factors - Science topic

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

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).

source: https://www.britannica.com/science/transcription-factor

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:

Thanks!!

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?

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?

I have a list of transcription factors and i'm aiming to find the genes that are regulated by these TFs

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

I'm looking for a bacterial promoter/transcription factor which can sense Acetyl-CoA.

Thank you in advance.

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?

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.

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?

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?

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.

I have a list of significant SNPs and would like to perform a Motif Discovery and Transcription Factor analysis.

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)?

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.

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.

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!!

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??

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.

I want to perform Luciferase assay to check the strength of the selected promoter and binding of the transcription factor.

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!

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?

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.

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.

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.

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

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?

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.

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! =)

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!

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.

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.

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!

For transcription factors co-locate with enhancer DNA sequences

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.

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.

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

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!

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.

Hi everyone,

I have a list of differentially expressed genes from which I want to :

Could you please suggest some tools/R packages or platforms to perform this kind of analysis?

Thank you.

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.

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..

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?

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?

I would like to know about all the molecules that can be expressed by/in CD8 T cells

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.

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

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.

Is there any database that can provide me with information on how a particular gene is regulated (for example, names of transcription factors)

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.

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.

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.

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?

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.

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????

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?

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!

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.

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,

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!

e.g., the level of the transcription factor "TP53" in Alzheimer's disease?

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  

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!

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?

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/

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?

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.

Actually, my question is: can the transcription factor bind to RNA?

Do you know a tool for finding transcription factors that bind to RNA?

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 !

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?

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.

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?

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.

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.

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

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

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!