Overview

  • Product name
  • Description
    Rabbit polyclonal to PAR4
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IP, ICC, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide corresponding to Human PAR4 aa 324-342.
    Sequence:

    EQLKQENKTLLKVVGQLTR


    (Peptide available as ab5853)

  • Positive control
    • Rat spleen extract. This antibody gave a positive result when used in the following formaldehyde fixed cell lines: A549

Properties

Applications

Our Abpromise guarantee covers the use of ab5787 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use a concentration of 2 µg/ml. Detects a band of approximately 40 kDa (predicted molecular weight: 41 kDa).Can be blocked with PAR4 peptide (ab5853).
IP Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration. PubMed: 19801652
ICC/IF Use a concentration of 5 µg/ml.

Target

  • Function
    Pro-apoptopic protein capable of selectively inducing apoptosis in cancer cells, sensitizing the cells to diverse apoptotic stimuli and causing regression of tumors in animal models. Induces apoptosis in certain cancer cells by activation of the Fas prodeath pathway and coparallel inhibition of NF-kappa-B transcriptional activity. Inhibits the transcriptional activation and augments the transcriptional repression mediated by WT1. Down-regulates the anti-apoptotic protein BCL2 via its interaction with WT1. Seems also to be a transcriptional repressor by itself. May be directly involved in regulating the amyloid precursor protein (APP) cleavage activity of BACE1.
  • Tissue specificity
    Widely expressed. Expression is elevated in various neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer, Parkinson and Huntington diseases and stroke. Down-regulated in several cancers.
  • Domain
    The leucine-zipper domain is not essential for apoptosis, but is required for sensitization of cells to exogenous apoptotic insults and for interaction with its partners.
    The SAC domain is a death-inducing domain selective for apoptosis induction in cancer cells. This domain is essential for nuclear entry, Fas activation, inhibition of NF-kappa-B activity and induction of apoptosis in cancer cells.
  • Post-translational
    modifications
    Preferentially phosphorylated at the Thr-163 by PKC in cancer cells.
  • Cellular localization
    Cytoplasm. Nucleus. Mainly cytoplasmic in absence of apoptosis signal and in normal cells. Nuclear in most cancer cell lines. Nuclear entry seems to be essential but not sufficient for apoptosis (By similarity). Nuclear localization includes nucleoplasm and PML nuclear bodies.
  • Information by UniProt
  • Database links
  • Alternative names
    • 2310001G03Rik antibody
    • PAR 4 antibody
    • PAR-4 antibody
    • Pawr antibody
    • PAWR_HUMAN antibody
    • PRKC Apoptosis WT1 Regulator antibody
    • PRKC apoptosis WT1 regulator protein antibody
    • Prostate apoptosis response 4 protein antibody
    • Prostate apoptosis response protein 4 antibody
    • prostate apoptosis response protein PAR-4 antibody
    • Transcriptional repressor Par-4-like protein PAWR antibody
    • Transcriptional repressor PAR4 antibody
    • WT1 Interacting Protein antibody
    see all

Images

  • ICC/IF image of ab5787 stained A549 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab5787 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Shows a Western blot of PAR4 on rat spleen extract using ab5787.

  • Immunohistochemical analysis of paraffin-embedded mouse embryonic corneal tissue, staining PAR4 with ab5787.

    Embryos were fixed with 4% paraformaldehyde and treated for antigen retrieval by boiling for 10 min in the citrate buffer (pH 6.0). Sections were incubated with primary antibody and a biotinylated anti-rabbit secondary antibody.

References

This product has been referenced in:
  • Acharya M  et al. A complex regulatory network of transcription factors critical for ocular development and disease. Hum Mol Genet 20:1610-24 (2011). IP . Read more (PubMed: 21282189) »
  • Fernandez-Marcos PJ  et al. Simultaneous inactivation of Par-4 and PTEN in vivo leads to synergistic NF-kappaB activation and invasive prostate carcinoma. Proc Natl Acad Sci U S A 106:12962-7 (2009). IHC-P ; Human . Read more (PubMed: 19470463) »
See all 4 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Vielen Dank für Ihre Nachricht.

Es tut mir leid, dass Sie Probleme mit diesem PAR4-Antikörper hatten. Wie besprochen, habe ich eine kostenlose Ersatzlieferung mit ab5787für Sie in Auftrag gegeben. Sie hat die Referenznummer x und solltenach dem 1. Maibei Ihnen ankommen.

Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

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Answer

Thanks for your call earlier this week and for your patience. The immunogenic peptide for the PAR4 antibody ab5787 is available as ab5853- https://www.abcam.com/PAR4-peptide-ab5853.html You can use this peptide in a blocking assay in order to demonstrate the specificity of the antibody, since there is not negative control tissue available. Please let me know if you have any questions or if there is anything else that I can do for you.

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Question

I have a complaint about the Par4 antibody AB5787 (Lot. 45056) which seems to bind unspecific in WB of HT22 celllysate. Our customer has reviewed your suggestions and done some additional experiments - unfortunately without success. Here are his answers to your questions: 1. This antibody has been characterized on human and rat samples for Western blot application. Other species have not been tested so we do not know if it recognizes mouse PAR4. It is expected to cross-react with mouse (100% identity with immunogen) due to sequence homology but we have no data yet. - "Now tried the antibody with proteins extracted from rat neurons. The result was as bad as before". 2. As I understand the customer has multiple bands. How much protein was loaded onto the gel? We usually recommend 20-30 ug total protein/lane. If the gel is overloaded, multiple bands can be seen. - "I loaded the gel several times with 50µg and several times with 25µg protein. At the lower loaded gel there also were mutliple bands". 3. Has the customer used protease inhibitors in the lysis buffer? No information in the e-mail. - "There was protease inhibitor cockatil in the lysis buffer". 4. It is also important to test the secondary antibody "run a secondary control" to see if the detection system works properly. Has he tested this? Or has he used the same secondary antibody with another primary antibody? What exactly the specification of the secondary antibody? - "The secondary antibody is polyclonal anti rabbit and it works great with other primary antibodies". 5. We would also recommend using positive control along with the samples. Rat tissue extracts, including testes, spleen, ovary, lung, and skeletal muscle would be good positive control. The customer should be able to see a 40 kDa and a 38 kDa protein bands. - "In cells transfected with Par4-Plasmid I cant see any bands at 40 or 38 kDa. Just the many other bands". Do you have any further suggestions?

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Answer

Thank you for your patience. In reviewing the information, we see Western blot with multiple bands at 40 kDa, 38 kDa and unknown at 20 kDa. It is most likely that the preparation, protocol or storage of antibody that is causing the extra bands. There may be homologous sequences in this protein because in reference papers, we noticed all the researchers have cut out the target band and did not reveal the entire blot. This occurs mostly when they want to avoid revealing multiple bands. We would suggest increasing the stringency of their washes and blocking (increase detergent/salt concentration, increase temperatures and times, while decreasing the concentration of primary antibody being used.) We would also recommend using positive controls, to show which bands are relevant. We hope this helps.

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