p53 - Science topic

I want to know whether p53 of ES2 cell line is mutant or WT?

I am using a 50bp DNA ladder. I have amplified p53 which was supposed to show at 166bp but is apparently at 500bp according to the image. I think the ladder is the problem, why is it starting so far from the well? This is a 2% agarose gel with 2ul EtBr. Voltage was 120 for 1 hour. I used 3ul of the ladder and 2ul dye. What am I doing wrong?

Dear all,

I'm using lentiviruses to knock down p53, and have had remarkable success so far with un-concentrated harvested media.

I have noticed that the transduction efficiency declines over the period of a few weeks storage at -80, and I was wondering if anyone has a suggested protocol for cryopreservation of lentiviruses.

I'm considering adding 0.5M sucrose and 0.6mg/ml BSA to the media as per Bandeira et. al.

Also, is there a freezing method which works best? Flash-freezing in liquid nitrogen, or ethanol/dry ice?

Thanks!

Sam

Bandeira V, Peixoto C, Rodrigues AF, Cruz PE, Alves PM, Coroadinha AS, Carrondo MJ. Downstream processing of lentiviral vectors: releasing bottlenecks. Hum Gene Ther Methods. 2012 Aug;23(4):255-63. doi: 10.1089/hgtb.2012.059. Epub 2012 Aug 30. PMID: 22934827.

In gnomAD website (https://gnomad.broadinstitute.org/), you can write a gene name (such as BRCA1 or P53) in the search box and you can obtain information for a subset of variants as a csv file.

I was wondering if there is a way to do this in a more programmatic way with Python, R or bash or any other API.

Can I get the paper related to p53 mutation and HS578T cell line.

In our experiment, we are checking and comparing two synthetic chemical compounds' impact on the expression levels of apoptotic and proto-oncogenic genes including p53, Bcl-2, cMyc, hTERT, and MMP13. But, we got an interesting and unexpected result. Our qPCR results showed that the expression of the P53 gene increased compared to the control, and the expression of the other genes also shows an increased expression level compared to the control. While we expect the expression of proto-oncogenetic genes (Bcl-2, cMyc, hTERT, and MMP13) to decrease.

Do you think there is a scientific explanation for this discrepancy?

Attached you can find our results.

Hello,

As a part of my research work, I need to detect the wild type p53 level in MCF-7 cells using western blotting. For this purpose i would like to know which antibody should i purchase (i.e) either monoclonal antibody or polyclonal antibody. If it is mnoclonal antibody, will it have any effect if it is produced by immunizing the animal with recombinant human p53 protein expressed in E.coli? Further, if there is any cat log no: available for the product (p53 antibody), i kindly request you to provide. Please do the needful.

Thanks in advance.

Hello everyone

I wanted to dock NQO1 and p53 (protein-protein docking).

NQO1 (a homodimer) is a quinone reductases which has FAD bound to both of its active sites. In order to catalyse the reduction of quinones (its substrate), the FAD within the active site has to be reduced to FADH2 by the oxidation of NADPH which then makes way for the incoming quinone (ping-pong mechanism).

NQO1 is also known to protect p53 from Ubiquitin independent 20S proteasomal degradations and so mutations affecting NQO1 could alter p53 stability (this is my study interest). In pursuit of investigating this association, I want to dock p53 with NQO1 homodimer (having both FAD and NADPH within the active sites).

Now here's the problem

In the absence of a crystallographic structure bearing both the ligands together (FAD and NADPH), I want to dock NADPH first to the NQO1 protein (protein-ligand docking). Being new to docking and after reading multiple threads, I realized that validating my docking protocol is essential.

To validate my docking protocol (done using AutoDock Vina), I considered a crystallographic complex protein (1kbq, https://www.rcsb.org/structure/1KBQ) with two ligands in it – namely FAD and 5-methoxy-1,2-dimethyl-3-(4-nitrophenoxymethyl)indole-4,7-dione and performed the docking of the same complex (I removed 5-methoxy-1,2-dimethyl-3-(4-nitrophenoxymethyl)indole-4,7-dione from the structure and used it as a ligand). Ligand and protein preparation was carried out using AutoDock Tools.

Please note – I tried docking using ligand obtained from two sources

1. Ligand from PubChem (didn’t yield appropriate pose even after energy minimization or reduction of active torsions, Structure labelled B in the figure attached)

2. Ligand derived from 1kbq itself (yielded appropriate pose after reduction of active torsions without energy minimization) (structure A and its derivatives)

As I have learned, to validate my docking, I need to compare RMSD between the crystallographic structure and my docked pose which should be below 2A (pardon me if I am wrong). Although my docking seems perfect (as evident from the last figure, after reducing 4 out of 5 active torsions for the ligand) I have failed to calculate the RMSD. I have tried online servers like COMPARE (https://webs.iiitd.edu.in/raghava/ pldbench/compare.php) and LS-align (https://zhanglab.ccmb.med.umich.edu/LS-align/) but no results. I have also tried comparing the RMSD using Discovery studio visualizer wherein I have used the original crystallographic structure bearing both the ligands as reference by selecting my target ligand in the structure (Structure>RMSD> Set reference) and compared it to my docked pose (out_ligand_1.pdbqt Structure>RMSD>All atoms/ Heavy atoms). The message prompted is

“The following molecule(s) failed due to the number of atoms not matching the reference ligand”

1. Please help me calculate the RMSD between the two structures.

2. Further, by the looks of it (as the docked pose superimposes the crystallographic structure) can I assume that my docking protocol is validated?

If yes,

3. Should I dock NADPH to both the active site one at a time (as there are two active sites within the homodimer) or both the active sites can be docked simultaneously (if yes, kindly guide me)?

4. If my docked NADPH exhibits interactions similar to other ligands (interactions mapped from reported NQO1 crystallographic structures), can I use this structure for protein-protein docking using online servers?

Thanking you for your patient reading.

I have some cell pellets that have been stored at -80C for a couple of months, and I'd like to make cell extracts of these to run in a human p53 ELISA.

Hello, I have one problem with the sequence of DNA which would be used as the evidence for lung cancer Please help me and guide me about this problem.

Thank you.

I have been working on recombinant p53R248Q and p53WT. It is necessary to show that the proteins are functional. For the wild type, I can show that the protein specifically binds to cognate DNA sequence. For p53R248 this doesn't hold true because the mutation abrogates DNA binding activity. So how do I show that p53R248 protein that we expressed and purified is what it is from a functional point of view. Is showing loss of function compared to the wild type protein enough?

What would be the best and fast way to determine if my recombinant p53 protein is functional?

Would like to get Remi Mito et al 2020 full article under the following title: Clinical impact of TROP2 in non‐small lung cancers and its correlation with abnormal p53 nuclear accumulation.

Many thanks

Can anyone recommend me a good mouse p53 antibody for Immunohistochemistry staining in human brain?

Thanks!

Hello

I've used Qupath to measure p53 expression in HeLa cells. Sometimes Qupath shows reasonable results (like brown cells detected as positive cells as they should be) and sometimes it doesn't. I've tried changing the stain set with ROI but the result still seems unreasonable. I want to ask everyone about this case, any answers and suggestions will help me a lot

Thank you in advance

Hi,

I'm a little bit confused about how to count the cell and measure the expression of p53 in immunocytochemistry method. Would someone recommend to me some software or web that I can use to analyze it?

Thank you

Hello,

I have consistently had problems with a variety of antibodies for p53 in Western blots (bands at incorrect heights / unspecific bands). I am trying to detect the level of p53 protein from mouse tissue samples. If anyone has any advice or antibody recommendations I would be really grateful to hear them!

Thanks,

Georgia

Hi, I'm currently doing a research about leaves extract and its potential to induced an apoptosis in HeLa cell, and I little bit confused about this case

increased expression of p53 by a drug will lead cell to induced an apoptosis, but what about the existence of HPV's E6 that is still present in the cell and maybe E6 protein and its pathways will inactivating some p53 that already expressed?

Thank you for sharing any answer to me !

I little bit confused about the p53 pathway in HeLa cell. It is about the HPV's infection. I had read some journals, but some of them explained that it was E6 protein that bind to p53 protein, so p53 protein can't work properly.

But, half of them explained that it was E6 gene that disturbed the transcription of p53 gene, and the cell can't produce p53 protein.

I am a beginner in this field and my knowledge is quite basic. So any answer will help me a lot. Thank you so much!

I have tried to express GST-p53 in E.coli (BL21-DE3, RIL, RIPL) but expression level is weak. I have come across few papers and protocols on google where researchers have purified p53 just with a His-tag. 

On troubleshooting I have concluded it may be due to p53 polymorphism. Here are the details of my protein construct: NCBI-genebank BAC16799.1 (CDS@GenBank: AB082923.1). It turns out to be different from Uniprot ID: P04637-1. My protein has 72R, 273H, 309S.

Please give your suggestions.

If you have successfully purified p53 in E.coli, please share your sequence ID.

Thank you.

I am currently using TCGA data available in cbioportal to perform analysis of different p53 targets. There's mRNA level data of all patients and all genes and about half of the patients have protein levels information for just some proteins. Both mRNA levels and protein levels are given in Z-scores. Since I was just getting started with the analysis and still becoming familiarized with everything, I decided to try and plot the p53 protein z-score vs the p21 mRNA z-score to see what it looked like, since p21 is a well-known p53 target. But to my surprise, the scatter plot looked quite random and uncorrelated (I attached below the image of the plot I did in R). I performed a correlation test between the p53 protein z-score and p21 mRNA z-score using Spearman's coefficient (since the distributions aren't normal) and got a very low number, around -0.002. I wanted to know if there's an error in the statistical methodology I'm applying and if not, what could be the explanation to this, since p53 has been widely proven to activate p21 so you would expect at least *some* degree of correlation.

Thanks in advance and sorry if it's a dumb question, I'm new to the field of databases and biostatisical analysis.

I am working on HT-29 and I am trying to figure out whether a certain compound disturbs the formation of grp75-p53 complex. I used a p53 mAb (D0-1) and protein A agarose beads from Santa cruz for the IP experiment. However, in the WB following IP, only p53 appeared and no GRP75 was obsevered.

What could be the problem? I froze and thawed my lysate for once, could it break the complex?

Or more fundamentally, the complex between grp75 and p53 just doesn't form in HT-29 cells?

"Severe acute respiratory syndrome coronavirus (SARS-CoV) is one of the most pathogenic human coronaviruses. Virulence is reflected in the molecular interplay between virus and host cells. Here we show a strategy of how SARS-CoV antagonizes the host antiviral factor p53, which impairs viral replication. The papain-like protease of the nonstructural protein 3 of SARS-CoV and other coronaviruses physically interact with and stabilize E3 ubiquitin ligase ring-finger and CHY zinc-finger domain-containing 1 (RCHY1), thereby augmenting RCHY1-mediated degradation of p53. The SARS-unique domain (SUD) enhances these effects. Knockout of p53 promotes replication of SARS-CoV replicons and of infectious virus. Taken together we identify cellular p53 as antiviral measure of coronavirus-infected cells, which is counteracted via the stabilization of RCHY1 by viral SUD and papain-like protease (PLpro) proteins and via ubiquitination of p53. "

https://www.pnas.org/content/113/35/E5192

Several research data showed that virus COVID-19 induce degradation of P53.

" the molecular mechanism of this inhibition remains unclear. Here, we provide evidence that PLP2, a catalytic domain of the nonstructural protein 3 of human coronavirus NL63 (HCoV-NL63), deubiquitinates and stabilizes the cellular oncoprotein MDM2 and induces the proteasomal degradation of p53 "

Is it meaning that in next years Word will meet spike of cancer and immunepathology diseases ?

p53 Degradation by a Coronavirus Papain-like Protease Suppresses Type I Interferon Signaling

Hi,

I'm knew to western blotting (with only some experience in FACS in the past). For my project we'd like to investigate the apoptosis machinery. I've started off with "easy" antibodies, looking at PARP cleavage, which went quite well.

For me to practice other antibodies; we cultured a p53 wt (SU-DHL-5) and a mt (MEC2) cell line with various concentrations of nutlin3a and assessed MDM2, p53 and p21 expression. If I understood the setup correctly, nutlin3a should lead to MDM2 depletion and correspondlingly to an increase in p53 and p21 levels (however, only in p53 wt cell lines).

I've cut the membrane into three parts, and incubated each with the corresponding primary antibody. beta-Actin as a loading control was stained in a second run after stripping.

I'm somehow unable to detect any signal with the MDM2 antibody. I re-did another gel, where I used a different clone and the result was the same. The experiment definitely worked in my lab in the past, I even ran a gel on some lysates students did a year ago, where it worked. But still same result.

I'm not sure where the issue lies; is it the lysates, my blotting or the stability of the antibodies (both clones are from different suppliers, but both state the antibody is only guaranteed to be stable for 12 months).

I'm also wondering a bit what I'm seeing with the p53 and p21 antibodies; For p53 I'm somehow detecting a second band at ≈ 35kDa and my p21 signal is somehow below the 20kDa marker.

Unfortunately, the transfer didn't go terribly well, so the blot I'm uploading isn't super pretty. But it was the third in a series of repeats, that all showed the same thing.

For me these proteins aren't crucial, as I'll mostly work with others. They were intended as a practice for me. I'm nonetheless posting this, as I'd like to understand what is going.

P.S. Are there any "comprehensive" guides on western blot wisdom, or is it just learning by doing?

Cheers, and all the best.

"The Poly(Lactic-co-glycolic acid) (PLGA) NPs: Poly(lactic-co-glycolic acid) (PLGA) NPs are the commonly used polymeric core NPs. This Food and Drug Administration (FDA)-approved polymeric core is biodegradable, biocompatible and non-toxic. These polymeric NPs, when encapsulated with some anticancer drugs or near-infrared (NIR) dye, allow them to be used in image–guided tumor therapy (Vijayan et al, 2018)."

"ONYX-015: selective replication in tumour cells with altered p53 pathway.

The striking similarities of tumour cells and adenovirus infected cells gave rise to the concept of using a mutant adenovirus to selectively eliminate tumour cells, which is based on the observation that in both, tumour cells and adenovirus infected cells, p53 – in its role as ‘guardian of the genome’ – is a major target for inactivation. ONYX-015 contains a 827 bp deletion in the E1B region of the viral genome and a point mutation that generates a premature stop codon preventing expression of a truncated form of the E1B55K protein (Barker and Berk, 1987). Theses mutations render ONYX-015 incapable of blocking p53's function. Indeed, dl1520 is incapable of degrading p53, which therefore accumulates in the nucleoplasm after infection of normal (p53+) cells. Infection of normal cells with ONYX-015 should evoke a p53 response: either growth arrest or apoptosis, resulting in aberrant viral replication. In contrast, tumour cells that do not possess a functional p53 gene (p53− cells) should support replication of ONYX-015. Replication of ONYX-015 should therefore be restricted to p53 deficient cells resulting in selective destruction of cancer cells (Rise et al, 2002)."

Ries, S., & Korn, W. M. (2002). ONYX-015: mechanisms of action and clinical potential of a replication-selective adenovirus. British Journal of Cancer, 86(1), 5–11. https://doi.org/10.1038/sj.bjc.6600006

Vijayan, V., Uthaman, S., & Park, I.-K. (2018). Cell Membrane-Camouflaged Nanoparticles: A Promising Biomimetic Strategy for Cancer Theragnostics. Polymers, 10(9), 983. https://doi.org/10.3390/polym10090983

Tumor suppressor genes (such as Rb, APC, PTEN and the ever famous p53) are often downregulated in cancer cells, if not completely lost or dysfunctional.

I'd like to know if there are any tumor suppressor genes that are upregulated in metastatic cancer cell lines/models? If so, could you link to articles/publications/studies of such genes? Are these genes bi-modal? Or have people figured out the molecular mechanism behind these genes?

I'd appreciate any and all help provided.

Thank you.

I have carried out a western blot to detect the p53 protein by using a primary and secondary antibody, then I have used RFLP with controls to detect differences between a mutated or normal wild type p53.

1. Accordingly to protein marker the proteins were at level 50kD, is that still fine or does it have to be at 53kD specifically to say its p53?

Other question included:

2. 'Would you expect a molecular weight difference from a single polymorphism? What about a 1 kb deletion or addition, how might that change the molecular weight of p53?'

- what is it supposed to mean?

-- i have detected that mutated had 3 bands whereas normal had 4, is this what the question is potentially refering to ? How can I describe the digestions in the RFLP?

Do I look at the most prominent bands on the figure? or do I look at bands at level 53kDa, even though they are not the most prominent in the image?

Would i also mention that multiple bands represent different p53 isoforms at that level?

Would appreciate the help :)

Also do i label it as molecular mass or Molecular weight if i am simply looking at a protein ladder of kDa?

Hi all

I performed a luciferase tagged PG13 wild type p53 promoter assay on p53 mutant (305ins4) HSC-3 cell line and found amplification of WT p53. Can anybody suggest how this could happen? Does the cell line have wild-type p53 allele too? Is there any study done on this?

Thank you so much in advance!

Just mechanisms exerting p53 gene to link chromosome are preceded p53 gene linking chromosome via viral affected cellular DNA subjected to accelerated cellular cycle due to stimulation G1, S, and G2 phases cellular cycle which induce also by mitochondrial processes and so on. (see bellow:

1. Ponizovskiy M.R., (2019), A newer method cancer treatment which is based on Link Rearrangement Operations of T cells, International journal of Cancer and Oncology, 6(2), 42 – 51, doi:10.15436/2377-0902.19.2546 ISSN:2377-0902.

2. Ponizovskiy M.R., (2013), Biophysical and biochemical transmutation of mitochondrial function in cancer genesis, Biochemistry & Analytical Biochemistry, Volume 2, Issue 3, 1 – 9, doi:10.4172/2161-1009.1000137. ).

Hello

I would like to know if there is some alternative for using cyclotherapy without the role of p53

Thanks

I wondered whether p53 interacts with Myc-tag (EQKLISEEDL)? I try to perform a Co-IP, p53 accumulates with desired and undesired Myc-tagged proteins though. I'd be glad for any suggestions or related information. Thanks in advance!

has anyone ever measured the concentration of p53 protein using UV-Vis. can p53 be complexed using methylene blue for example?

I'm currently trying to label human p53 with small organic dyes (Alexa and/or Atto dyes). Does anyone have experience with labeling p53 with these dyes, and maybe would be able to suggest the best approach? I think one common way is to link these to Thiols, but I noticed a lot of cysteines are in the DNA binding domain and this might affect activity.

I transfected cells with three different plasmids expressing p53 gene. I also had non-transfected cells. I performed western blot of the whole-cell lysates and probed for p53 using DO-1 antibody. In all 4 conditions, I detected a band at approx ~53 kDa which is expected as all samples contain wild-type p53. But also I detected a band of ~58 kDa seen only with the three transfected conditions?

Does anyone have an idea of what that band represents?

Many thanks in advance

How are the levels of p53 monitored ? what techniques are used to study the stability of p53 during the cell cycle?

Hi everyone!

I want to study potential interactions between a protein of interest and p53. I read that false positive results are often observed with PPI involving p53. Is it necessary to provoke a stress in my cells to stabilized p53 to have more sensitive results? How can I be sure of my results?

Thanks for your help!

Marie

I generated these RKO Cas9 stable cell line to knock out p53 using different sgRNA (one at a time). To do that, I need to induce the expression of Cas9 with Doxycyclin. I have been using 4ug/ml for 7 days. On my polyclonal population, I see a slight reduction of p53 protein using WB. However, when I check my clones, there is no knockout, only knockdown as observed in the polyclonal cells.

I was wondering if I increase the induction time and concentration of Doxy could help me generate these knockouts. I know that if Doxy is too high, it can kill my cells, but I can't think of any other solution at this time. Will my chances of getting off-target effects from Cas9 increase? Any thoughts? Thanks, guys!

Hello everyone,

In actually I am new in this concept. I want to create plasmid system for dual luciferase assay(promega) for spesisfic gene and mutant version of it. For example P53 and several mutation on it. I want to see the effect of mutation on transcriptional activitiy.

For this what kind of insert can I use, how can I design it. I have some gaps in my mind. Anyone can suggest to me same paper or rewiev about it ?

Thank you.

Hi All

How long does it take to silence the p53 gene in breast cancer cells? Does the WT or mutant p53 status affect the transfection efficiency? Any other inforrmation regarding transfection wiuld be useful

Thanks for your time!!

Hi, everyone. Recently, I did the Y1H experiment (my first time) which gave me several problems and also had some issues made me confused. If you have any experience, hope you can give me some suggestion. Thanks very much in advance.

I used the Matchmaker Gold yeast one-hybrid system from Clontech.

The following are my problems and concerns:

I am sorryfor a quite long question. But I really need your help. I am looking forward receiving your kindness answers. Thanks all of you with my best wishes.

Role of MDM2 and other E3 ligases in the regulation of p53 ubiquitination and degradation have been deeply investigated. MDM2-p53 binding is well defined! I am interested to know whether p53 hotspot mutations either effect the MDM-p53 interaction or the ability of mutant p53 for K48-likned poly-ubiquitination. Other possibility would be only specific structurally distorted mutants are neutral for MDM2 actions. I am interested to find list of p53 mutants, which can still bind to the MDM2 and normally ubiquitinated. Similarly a list of mutant which can not bind to mutant p53 and its ubiquitination. I am searching for paper and/or review article which can provide me detail information of MDM2-mutant p53 interaction and Ubiquitination. Looking forward for you suggestions!

Hi

I am getting a strange result. With the upregulation of p53 one of the protein involved in metastasis is going up as well. When i treated the siRNA against p53 the level of p53 went down and again the levels of my gene (or protein) of interst also went down. Does anyone have explanation for this. To me it is very strange that why would p53 positively regulate the prometastatic protein?

I am attempting to devise a preliminary cell based assay that concerns the impact of SASP factors on uptake of a particular extracellular factor. The assay of uptake already works, however it uses a cell line, specifically the H4 (https://www.atcc.org/products/all/HTB-148.aspx#documentation) glioblastoma line, which has deletions of PTEN and CDK2A.

Since these cells don't have mutations in p53, they should still be able to be induced into senescence via irradiation, but I'm unsure if the SASP pathways will be intact due to those deletions. If they would prevent normal SASP expression, is there any (neuronal) cell line that would be suitable, or are my only options primary cells/iPSC derived cells?

Do MUT p53 protein really shuttles between nucleus and cytoplasm in absence of any genotoxic or metabolic stress?

I need these cell lines for my master thesis.

I have seen in some studies that the level of wild type p53 has increased after treatment with DNA damaging agents such as Doxorubicin in MDA-MB-231 . So is there any studies which show the amount of wild type or atleast confirms the presence of wild type p53 along with mutated p53 in MDA-MB-231?

To assess whether expression of the gene of our interest is associated with mutant status of p53 we have treated FaDu cells( mut-p53) with CP-31398, a Known p53 stabilizer, that restore a wild-type DNA-binding conformation of mutant p53. we check the expression by TaqMan based q-PCR, and got expected result, but when we treated the Fadu cells with Pifithrin-α( an inhibitor of p53 ) we again got the same trend( in both cases RQ is 0.2 & 0.4 respectively), while we are expecting No change or Induction. According to the publication: Cancer Biol Ther. 2002 Jan-Feb;1(1):47-55, FaDu expressed only 50% of the normal level of p53 mRNA, either because only one allele was present (A431), or because only one of the two alleles was transcribed. What could be the possible reason of same trend of gene expression after the exposure of CP-31398 & Pifithrin-α in Fadu Cells?

P53 (tumor suppressor gene) or marker.

Can detect the mutation of p53 by IHC (monoclonal Ab) with out use PCR

I have treated HeLa cells (a p53-null cell line) in order to increases its intracellular concentration of p53. Does anyone know many cells would be sufficient for a significant change in the ELISA data?

Actually I want to treat my cells in 6-well plates (~200,000 cells per well). So how many wells I must use for each of my conditions?

Hi

For how much time should cells be incubated with DNA damaging agent(s) like Camptothecin, etoposides so as to get expression of p53 and p21 (At RNA as well as protein level). Any image from a research article and the literature in this would be huge help.

THANKS!!

p53 is considered as the Guardian of the cell and suppresses tumorigenic genomic rearrangements. I want to learn more that the exact role of p53 in repairing faulty DNA repair mechanism in vivo following transient cell cycle arrest. Any specific sources/articles/papers and materials on this topic would be much appreciated.

I have 4 groups of treatment and for each group of treatment I calculated the percentage of P53 positive cells in 6 samples exposed to the treatment. So I have a 4x6 table.

I want to know if a certain treatment is giving me more P53 positive cells.

Is Chi-square test appropriate in this situation? Or are there alternatives? How do I get to know between which treatment I have the significancy?

Thanks.

I have extracted RNA from tumor tissue sample and wondering whether the basal level of p53 expression enough to do RT PCR amplification using p53 transcript specific primers?

Thank you

Mutant kras leads to constitutive activation of downstream factors, what if we directly delete Kras in the tumor tissue? Will there be tumor regression? Also, people don't target KRAS is because that KRAS is important in normal tissue? So the goal is to find genes that are important in KRAS signaling but not important in normal tissue? 

I work on Bioinformatics system for mutation detection in TP53, I would like to know the possible number of mutation per a codon i.e.:

Codon 248 consist of three bases of amino acid which is:

CGG

the mutation can be in only one base? such as:

CGG ---> CGC

or

CGG ---> GGG

and so on

Is it possible to have Mutation in two bases or more?

CGG ---> GCG

CGG ---> CAA

what will happen if the Mutation occurred in the three bases?

CGG ---> GCC

CGG --->TAC

Recent data show that elephants have billions more cells than humans while less than 5% of these giant animals die of cancer, compared to 25% of humans. Elephants have dozens of copies of the p53 gene compared to two copies in the human genome (thttps://doi.org/10.1016/j.celrep.2018.07.042)

How to use this knowledge in the fight against cancer?

A colleague who works with p53 needs to quantify this protein by Western Blot. He tried it in several ways, but he never could see the protein in the membrane. After checking for many possible protocol errors and antibody problems, he stained the gel with coumassie and did not check any band on the gel except once with the approximate size of p53. How can this happen?

I need to detect p53 in murine E15.5 embryos that overexpress this protein. The embryos were collected and fixed overnight in 10% Neutral Buffered Formalin. Then, I cryopreserved these embryos in 30% sucrose until they sunk to the bottom of the tube and embedded them in OCT. I took sections around 14um in positive charge slides. These slides were stored at -80C. I'm trying to use these slides for p53 IHC using CM5 antibody but no p53 has been detected. Does anyone has protocol for p53 IHC in frozen sections? Thank you in advance!

Dear Researchers,

I need a comprehensive guideline to follow to determine analysis cut-off points (e.g.: number of nuclei per sample, percentages to consider a deletion present, false +/- testing for each probe since the % of hybridisation is different for each probe).

I am using Leica: TP53, LEU1, +12 and ATM mutations for my FISH experiments. So far the only one I have found that is of any use is this:

Does anyone have any other papers that may be useful/helpful for my purpose? Would be better if it was specific to CLL.

Thanks in advance.

I'm trying to find an immortalized cell line of normal keratinocytes for UV irradiation and subsequent p53 studies...So shouln't be HPV transformed or SV40 transformed. I found NIKS...however I don't now how can I buy it. Thank you.

I have had success using DO-1 and higher dilutions of Pab240 (lower dilutions yield non-specific cytoplasmic signal) to detect nuclear p53, and I have read that DO-7 is another reliable antibody for nuclear p53 detection. Has anyone had reproducible success with antibodies to detect cytoplasmic p53 in IF experiments? I have read that BP53-12 (sc-263) could potentially be a good candidate - can anyone vet this?

I need a tumor cell line Cx43-deficient as negative control for a FACS/WB/IF detection of Cx43

Hello! I would like to design primers for a set of genes involved in apoptosis (p53, BAX, BCL2, etc) using chinese hamster CHO-K1 cell line. However, when I browse UCSC looking for the genes' sequence I find two different genomes: CHO K1 cell line/criGriChoV1 and C_griseus_v1.0/criGri1. The former refers to the actual cell line, whereas the latter refers to the hamster itself.

Which one do you think I should use for designing my primers?

Thanks in advance!

Gain-of-functional mutant p53 exhibits a longer half-life period as compared with wild-type p53, which is why the significant accumulation of the mutant p53 in cancer cells is recognized in many kinds of malignant neoplasms.

As compared with CEA and CA19-9, anti-p53 antibody (IgG subtype) can be detected in the very early stage of esophageal, colon, breast, prostate carcinomas etc.

Given that pseudo-positive ratio of anti-p53 antibody is less than 5% in the healthy population, the detection of anti-mutant p53 IgG antibody holds much promising.

I would like to know your opinion on this useful marker.

Thank you in advance!

Hello everyone,

In my project, I found that there were a subset of leukemia cell lines died if you knock out Fanconi anemia complex genes (e.g. FANCL, FANCT, FANCD2 etc), while some other leukemia cell lines have no effect. The leukemia cell lines in my experiment are all p53 mutant cell lines, so it is probably not because of the p53-induced apoptosis, there might be some other mechanism underlying this. What kind of mechanism it might be? (any link to homologous recombination? or anything else?) What experiment should I do to find out the molecular mechanism that underline these two different phenotype?

Hello everyone,

In my project, I found that there were a subset of leukemia cell lines died if you knock out Fanconi anemia complex genes (e.g. FANCL, FANCT, FANCD2 etc), while some other leukemia cell lines have no effect. The leukemia cell lines in my experiment are all p53 mutant cell lines, so it is probably not because of the p53-induced apoptosis, there might be some other mechanism underlying this. What kind of mechanism it might be? (any link to homologous recombination? or anything else?) What experiment should I do to find out the molecular mechanism that underline these two different phenotype. Thank you very much.

It is well known that P53 causes cell cycle arrest in G1-S and also on G2-M transition. But is there any evidence that P53 is responsible for S-G2 arrest? I have got only 1 paper saying that P53 is responsible for S-G2 arrest but there is no reference. If you know of any such papers please cite the ref.

Oncogene have an operator gene and tumor suppressor gene. The tumor suppressor gene is always turn-on and an operator gene is turn-off. There are several factors to create tumor suppressor to turn of by a mutation in the P53 protein to play oncogenesis.

Western blot image without ladder

Which strategy to adopt for amplifying full length wild-type p53 gene from human DNA?

BXPC-3 is p53 non-hotspot mutated Y220C cell line. In general, I wanted to ask which cell culture media to be used and how is its growth statistics?

I know that chromosomal instability is responsible for triggering cellular death in M2, but what pathway or signal is activated to connect the DNA damage and subsequently trigger apoptosis? Does p53 play any role in inducing apoptosis when cells reach M2? Or do other tumor suppressors play a role in inducing apoptosis at this state? Thanks.

Why longer p53 truncated proteins go undetected by IHC, even though DO7 antibody binds to the N- terminal of the p53 protein?

Identification and quantification of senescent cells in human tissues is a challenging task. It is well known that the application of a single biomarker such as SA-b-gal activity (the most widely used senescent marker) is not enough. Today we can chose among a large variety of senescent markers, such as gammaH2AX, Ki67, BrdU, HGMB1, p16INK4A, p15INK4B, p21CIP1, p53, DEC1, DCR2, B2MG and PAI1.

What combination will be the most efficient and cost effective?

Hi all,

I am trying to find the p53 mutation status of the HG3 CLL cell line through the most commonly used databases and papers available and had no luck so far.

Would anyone know whether this particular cell line expresses a wild type or mutated p53?

Many Thanks,

James

Hello all,

I know that the p53 gene is wild-type in MCF7, but are there any other mechanisms in place that suppress p53 function in MCF7? Or is p53 fully functional and ready to be activated in MCF7? Thanks.

Also, must all cancer cells have mutant or suppressed p53? Or are there any cancer cells that remain malignant despite having wild type p53 that is not surpressed?

Is MX-1 breast cancer cell line P53 wild type or mutant?

Are there any specific p53 antigen to specific antibodies or can we simply use p53 protein and detect it by DO-1 (p53 antibody ) ?