RIPK3/RIP3 Antibody

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - Analysis of RIP3 in HeLa cell lysate with RIPK3/RIP3 antibody [NBP1-77299] at (A) 1 and (B) 2 ug/mL. Observed molecular weight ~60 kDa. Theoretical molecular weight 53 kDa.

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - Western Blot Validation in Mouse Cell lines. 15 ug of lysates per lane.Antibodies: RIPK3/RIP3 Antibody [NBP1-77299], (0.5 ug/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Observed molecular weight ~50 kDa. Theoretical molecular weight 53 kDa.

Immunohistochemistry-Paraffin: RIPK3/RIP3 Antibody [NBP1-77299] - Immunohistochemistry analysis of a paraffin embedded mouse kidney tissue section using RIPK3/RIP3 Antibody [NBP1-77299] at 2.5 ug/mL concentration.

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - Analysis using RIPK3/RIP3 antibody [NBP1-77299]. L929 cell line (mouse). 20 ug whole cell lysate. Primary antibody: 1:2000. Secondary antibody: 1:20000. Western blot image submitted by a verified customer review. Observed molecular weight ~57 kDa. Theoretical molecular weight 53 kDa.

Immunocytochemistry/Immunofluorescence: RIPK3/RIP3 Antibody [NBP1-77299] - ICC/IF analysis of Rat's kidney cell culture using RIPK3/RIP3 Antibody [NBP1-77299] at 20 ug/mL concentration.

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - Western Blot Validation in C2C12 Cells. 15 ug of lysates per lane. Antibodies: RIPK3/RIP3 antibody [NBP1-77299], 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Lane 1: NBP1-77299, 0.1 ug/mL in the presence of peptide blocking. Lane 2: NBP1-77299, 0.1 ug/mL Lane 3: NBP1-77299, 0.2 ug/mL Lane 4: NBP1-77299, 0.5 ug/mL. Observed molecular weight ~55 kDa. Theoretical molecular weight 53 kDa.

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - Western Blot Validation in Human Cell Lines. 15 ug of lysates per lane. Antibodies: RIPK3/RIP3 antibody [NBP1-77299], (0.5 ug/mL), 1h incubation at RT in 5% NFDM/TBST. Secondary: Goat anti-rabbit IgG HRP conjugate at 1:10000 dilution. Observed molecular weight ~ 57 kDa. Theoretical molecular weight 53 kDa.

Immunohistochemistry-Paraffin: RIPK3/RIP3 Antibody [NBP1-77299] - Rat kidney tissue with RIPK3/RIP3 Antibody [NBP1-77299] at 5 ug/mL.

• Reactivity: Hu, Mu, Rt
• Applications: WB, ELISA, ICC/IF, IHC, IHC-P, IP, GS, KD, KO
• Clonality: Polyclonal
• Host: Rabbit
• Conjugate: Unconjugated
• Concentration: 1 mg/ml

RIPK3/RIP3 Antibody Summary

• Immunogen: RIPK3/RIP3 Antibody was made to a 14 amino acid peptide near the carboxy terminus of murine RIP3. The immunogen is located within the last 50 amino acids of RIP3. Amino Acid Squence: AQFGRGRGWQPFHK
• Specificity: Mouse RIPK3/RIP3 Antibody has one isoform (486aa, 53 kDa). Human RIP3 has 3 isoforms, including isoform 1 (518aa, 57 kDa), isoform 2 (252aa, 28 kDa) and isoform 3 (231aa, 25 kDa). Rat RIP3 also has one isoform (478aa, 52 kDa). NBP1-77299 can detect can detect isoforms of mouse and rat as well as human isoform 1.
• Isotype: IgG
• Clonality: Polyclonal
• Host: Rabbit
• Gene: RIPK3
• Purity: Peptide affinity purified

Applications/Dilutions

• Dilutions:
  • Western Blot 0.1 - 0.5 ug/mL
  • ELISA 1:100 - 1:2000
  • Immunocytochemistry/Immunofluorescence 20 ug/mL
  • Immunohistochemistry 5 ug/mL
  • Immunohistochemistry-Paraffin 5 ug/mL
  • Immunoprecipitation 20 ug/mL
  • Gel Supershift Assay
  • Knockdown Validated
  • Knockout Validated
• Application Notes: Gel supershift assay reported in scientific literature [PMID: 27721066]. Immunoprecipitation reported in scientific literature [PMID: 27861127]. RIPK3/RIP3 antibody validated for WB from a verified customer review. Knockout validation reported in scientific literature [PMID: 32246911]. Knockdown validation reported in scientific literature [PMID: 31655343]. ICC/IF, Immunohistochemistry, and Western Blot reported in multiple pieces of scientific literature.
• Theoretical MW: 53 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• Control Peptide:

  RIPK3/RIP3 Protein (NBP1-77299PEP)

Reactivity Notes

Human, mouse and rat reactivity reported in multiple pieces of scientific literature.

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS
• Preservative: 0.02% Sodium Azide
• Concentration: 1 mg/ml
• Purity: Peptide affinity purified

Alternate Names for RIPK3/RIP3 Antibody

• EC 2.7.11.1
• Receptor Interacting Serine/Threonine Kinase 3
• Receptor-interacting protein 3
• receptor-interacting serine/threonine-protein kinase 3
• receptor-interacting serine-threonine kinase 3
• RIP3
• RIP-3
• RIPK3
• RIP-like protein kinase 3

Background

RIPK3, Receptor interacting serine/threonine kinase 3 or RIP-like protein kinase 3 (human RIPK3 isoform1 theoretical molecular weight 57kDa) is a cytosolic protein with an amino terminal active kinase domain. RIP kinases, RIPK1 and RIPK3 play a central role in the induction of necroptosis, a form of programmed and inflammatory cell death that is caspase-independent (1). Necroptosis is a form of programmed necrosis which is initiated through the activation of RIPK3 by various ligands such as Fas, LPS and TNF. Formation of the necrosome results from the interaction between RIPK1 and RIPK3 mediated through their RIP homotypic interaction motifs (RHIMs) (1, 2). Following necrosome formation, RIPK3 activation leads to the phosphorylation of mixed lineage kinase domain-like protein (MLKL) which induces its oligomerization and translocation to the cell membrane where it alters plasma membrane integrity (2). Loss of membrane integrity results in lytic cell death, release of damage associated molecular patterns (DAMPs) and inflammation (2, 3). Therefore, the interaction of RIPK3 with other RHIM proteins is necessary for the initiation of necroptosis, while the execution phase is dependent on the activation of MLKL (4).

RIPK3 has several phosphorylation sites that are required for its role in necroptosis. For example, serine204 in mouse, which is conserved in the human (serine199), is necessary for necroptosis while serine residues 232 and 227 are both required for RIPK3s interaction with MLKL (2). The core necroptosis proteins RIPK1/RIPK3 and MLKL are implicated in several disease states such as neurodegeneration, cardiovascular, hepatic and pulmonary disease (3).

References

1. Orozco, S., & Oberst, A. (2017). RIPK3 in cell death and inflammation: the good, the bad, and the ugly. Immunological Reviews. https://doi.org/10.1111/imr.12536

2. Dhuriya, Y. K., & Sharma, D. (2018). Necroptosis: A regulated inflammatory mode of cell death. Journal of Neuroinflammation. https://doi.org/10.1186/s12974-018-1235-0

3. Choi, M. E., Price, D. R., Ryter, S. W., & Choi, A. M. K. (2019). Necroptosis: A crucial pathogenic mediator of human disease. JCI Insight. https://doi.org/10.1172/jci.insight.128834

4. Lee, K.-H., & Kang, T.-B. (2019). The Molecular Links between Cell Death and Inflammasome. Cells. https://doi.org/10.3390/cells8091057

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for RIPK3/RIP3 Antibody (NBP1-77299)(46)

Publications using NBP1-77299

• Dai Q, Zhang Y, Liao X et al. Fluorofenidone Alleviates Renal Fibrosis by Inhibiting Necroptosis Through RIPK3/MLKL Pathway Frontiers in pharmacology Dec 16 2020 [PMID: 33390935] (WB, Mouse)
• LingHu HR, Luo H, Gang L Bufalin Induces Glioma Cell Death by Apoptosis or Necroptosis Onco Targets Ther May 27 2020 [PMID: 32581545] (WB, IP, Human)
• Seo J, Seong D, Nam YW et al. Beclin 1 functions as a negative modulator of MLKL oligomerisation by integrating into the necrosome complex Cell Death Differ. May 26 2020 [PMID: 32457484] (WB, Human, Mouse)
• Lyu AR, Kim TH, Park SJ et al. Mitochondrial Damage and Necroptosis in Aging Cochlea Int J Mol Sci Apr 3 2020 [PMID: 32260310] (IHC, Mouse)
• Zhou H, Xu C, Lee H et al. Berardinelli-Seip congenital lipodystrophy 2/SEIPIN determines brown adipose tissue maintenance and thermogenic programing Mol Metab Mar 4 2020 [PMID: 32246911] (WB, KO, Mouse)
• Colijn S, Muthukumar V, Xie J et al. Cell-specific and athero-protective roles for RIPK3 in a murine model of atherosclerosis Dis Model Mech Jan 17 2020 [PMID: 31953345] (WB, Mouse)
• Popper B, Rammer MT, Gasparitsch M, et al. Neonatal obstructive nephropathy induces necroptosis and necroinflammation Sci Rep Dec 9 2019 [PMID: 31819111] (WB, Mouse)
• Du XK, Ge WY, Jing R, Pan LH Necroptosis in pulmonary macrophages mediates lipopolysaccharide-induced lung inflammatory injury by activating ZBP-1 Int. Immunopharmacol. 2019 Oct 23 [PMID: 31655343] (WB, KD, Mouse)
• Takezaki A, Tsukumo S I et al. A homozygous SFTPA1 mutation drives necroptosis of type II alveolar epithelial cells in patients with idiopathic pulmonary fibrosis. J Exp Med Feb 12 2019 [PMID: 31601679] (IP, Mouse)
• RIPK3 Activation Leads to Cytokine Synthesis That Continues After Loss of Cell Membrane Integrity Orozco SL, Daniels BP, Yatim N et al. Cell Rep [PMID: 31461645] (WB, Mouse)
• The Neuroprotective Effects of Necrostatin-1 on Subarachnoid Hemorrhage in Rats Are Possibly Mediated by Preventing BloodBrain Barrier Disruption and RIP3-Mediated Necroptosis Chen J, Jin H, Xu H et al. Cell Transplant. [PMID: 31370690] (WB, Rat)
• Lim J, Park H, Heisler J et al. Autophagy regulates inflammatory programmed cell death via turnover of RHIM-domain proteins Elife Jul 9 2019 [PMID: 31287416] (WB, Mouse)
• Lloyd AF, Davies CL, Holloway RK et al. Central nervous system regeneration is driven by microglia necroptosis and repopulation Nat. Neurosci. Jul 1 2019 [PMID: 31182869]
• Font-Belmonte E, Ugidos IF, Santos-Galdiano M et al. Post-ischemic salubrinal administration reduces necroptosis in a rat model of global cerebral ischemia J. Neurochem. Jun 5 2019 [PMID: 31165478] (Rat)
• Li X, Gong W, Wang H et al. O-GlcNAc Transferase Suppresses Inflammation and Necroptosis by Targeting Receptor-Interacting Serine/Threonine-Protein Kinase 3 Immunity Feb 5 2019 [PMID: 30770249] (WB, Human)
• Hernandez D E, Salvadores N A et al. Axonal degeneration induced by glutamate excitotoxicity is mediated by necroptosis. J Cell Sci Nov 19 2018 [PMID: 30333135] (WB, Rat)
• Hou H, Wang Y, Li Q et al. The role of RIP3 in cardiomyocyte necrosis induced by mitochondrial damage of myocardial ischemia-reperfusion. Acta Biochim. Biophys. Sin. (Shanghai) Sep 12 2018 [PMID: 30215665] (WB, Rat)
• Generation and Use of Chimeric RIP Kinase Molecules to Study Necroptosis Rodriguez DA, Green DR. Methods Mol Biol. [PMID: 30136231] (WB, Mouse)
• Chen T, Pan H, Li J et al. Inhibiting of RIPK3 attenuates early brain injury following subarachnoid hemorrhage: Possibly through alleviating necroptosis Biomed. Pharmacother. Aug 13 2018 [PMID: 30114640] (ICC/IF, WB, Rat)
• Li P, Zhou Y, Goodwin AJ et al. Fli-1 Governs Pericyte Dysfunction in a Murine Model of Sepsis. J. Infect. Dis. Jul 20 2018 [PMID: 30053030] (WB, Mouse)
• Zamaraev AV, kopeina GS, Buchbinder JH et al. Caspase-2 is a negative regulator of necroptosis. Int. J. Biochem. Cell Biol. Jul 17 2018 [PMID: 30025878] (WB, Human)
• Piao X, Miura R, Miyake S et al. Blockade of TNF receptor superfamily 1 (TNFR1)-dependent and TNFR1-independent cell death is crucial for normal epidermal differentiation. J. Allergy Clin. Immunol. Mar 27 2018 [PMID: 29596938] (Mouse)
• Feldmann Friederike, Schenk Barbara, Martens Sofie et al. Sorafenib inhibits therapeutic induction of necroptosis in acute leukemia cells. Oncotarget 2017 [PMID: 28978109] (WB, Human)
• Martens S, Jeong M, Tonnus W et al. Sorafenib tosylate inhibits directly necrosome complex formation and protects in mouse models of inflammation and tissue injury Cell Death Dis 2017 Jun 29 [PMID: 28661484] (WB, Mouse)
• Sridharan H, Ragan KB, Guo H et al. Murine cytomegalovirus IE3-dependent transcription is required for DAI/ZBP1-mediated necroptosis EMBO Rep. 2017 Jun 12 [PMID: 28607035] (Mouse)
• Gong YN, Guy C, Olauson H et al. ESCRT-III Acts Downstream of MLKL to Regulate Necroptotic Cell Death and Its Consequences. Cell. Apr 6 2017 [PMID: 28388412] (Mouse)
• Ros U, Pena-Blanco A, Hanggi K et al. Necroptosis Execution Is Mediated by Plasma Membrane Nanopores Independent of Calcium. Cell Rep 2017 Apr 04 [PMID: 28380356] (WB, Mouse)
• Yang SH, Lee DK, Shin J et al. Nec-1 alleviates cognitive impairment with reduction of Abeta and tau abnormalities in APP/PS1 mice. EMBO Mol Med. Nov 17 2016 [PMID: 27861127] (IP, WB, Mouse)
• Shindo R, Yamazaki S, Ohmuraya M et al. Short form FLICE-inhibitory protein promotes TNFalpha-induced necroptosis in fibroblasts derived from CFLARs transgenic mice. Biochem. Biophys. Res. Commun. Nov 4 2016 [PMID: 27721066] (GS, Mouse)
• Qin D, Wang X, Li Y et al. MiR-223-5p and -3p Cooperatively Suppress Necroptosis in Ischemic/Reperfused Hearts. J. Biol. Chem. Aug 8 2016 [PMID: 27502281] (WB, Mouse)
• Lloyd AF Cellular and molecular mechanisms underpinning microglial activation during remyelination Thesis [PMID: 27446913] (ICC/IF, IHC, Mouse, Rat, Human)
• Seo J, Lee EW, Sung H et al. CHIP controls necroptosis through ubiquitylation- and lysosome-dependent degradation of RIPK3 Nat. Cell Biol. Mar 1 2016 [PMID: 26900751] (WB, IP, Human, Mouse)
• Wertz IE, Newton K, Seshasayee D et al. Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation. Nature. 2015 Dec 17 [PMID: 26649818] (WB, Mouse)
• Yoon S, Bogdanov K, Kovalenko A, Wallach D et al. Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it Cell Death Differ. 2015 Jul 17 [PMID: 26184911] (WB, Mouse)
• Miki Y, Akimoto J, Moritake K et al. Photodynamic therapy using talaporfin sodium induces concentration-dependent programmed necroptosis in human glioblastoma T98G cells Lasers Med Sci. 2015 Jun 25 [PMID: 26109138] (WB, Human)
  
  Details:
  human glioblastoma multiforma tumor cell line T98G treated with Control siRNA or (Fig. 4)
• Dara L, Johnson H, Suda J et al. Receptor Interacting protein kinase-1 mediates murine acetaminophen toxicity independent of the necrosome and not through necroptosis Hepatology. 2015 Jun 16 [PMID: 26077809] (WB, Mouse)
• Preyat N, Rossi M, Kers J et al. Intracellular nicotinamide adenine dinucleotide promotes TNF-induced necroptosis in a sirtuin-dependent manner. Cell Death Differ. Jan 1 2016 [PMID: 26001219] (WB, Human)
• Gautheron J, Vucur M, Reisinger F et al. A positive feedback loop between RIP3 and JNK controls non-alcoholic steatohepatitis. EMBO Mol Med. 2014 Jun 24 [PMID: 24963148] (WB, Mouse)
  
  Details:
  Figs 1B, S1, S11: RIPK3-/- and WT liver, Fig S10C: L929 fibrosarcoma cells. RIPK3 was detected between the 50 and 60 kDa marker. The specificity of the RIPK3 pAb was validated using RIPK3-/- and WT mouse liver by WB (Figs 1B, S11).
• Orozco S, Yatim N, Werner MR et al. RIPK1 both positively and negatively regulates RIPK3 oligomerization and necroptosis. Cell Death Differ. 2014 Jun 06 [PMID: 24902904] (WB, Mouse)
  
  Details:
  Figs 4b, S1B, S1I: Jaxson immortalized fibroblasts (Jax) and NIH3T3 cells transfected with RIPK3 fusion constructs (RIPK3-NIH3T3). RIPK3 was detected at ~50 kDa in Jax and at ~70 kDa in RIPK3-NIH3T3 cells. The specificity of the RIPK3 pAb was validated in RIPK3-NIH3T3 cells by WB (Figs 4b, S1B, S1I). Note: According to the authors, NIH3T3 cells do not expression endogenous RIPK3.
• Berger SB, Kasparcova V, Hoffman S et al. Cutting Edge: RIP1 kinase Activity Is Dispensable for Normal Development but Is a key Regulator of Inflammation in SHARPIN-Deficient Mice. J. Immunol. 2014 May 12 [PMID: 24821972] (WB, Mouse)
  
  Details:
  Bone marrow-derived macrophages, Fig 1C.
• Dillon CP, Weinlich R, Rodriguez DA et al. RIPk1 Blocks Early Postnatal Lethality Mediated by Caspase-8 and RIPk3. Cell. 2014 May 22 [PMID: 24813850] (WB, Mouse)
  
  Details:
  Mouse embryo fibroblasts, Figs 6D, 6H, S4D, S6A, S6D.
• Piao X, Komazawa-Sakon S, Nishina T et al. c-FLIP maintains tissue homeostasis by preventing apoptosis and programmed necrosis. Sci Signal. 2012 Dec 18 [PMID: 23250397] (WB)
  
  Details:
  WB: Fig 1B: primary RIPK3 knockout and wildtype mouse embryo fibroblasts (MEF) and 3T3-SA cells. Fig 2F: 3T3-SA Fig S1D: various tissues from RIPK3 knockout and wildtype mice, 3T3-SA Fig S3C: SVEC cells expressing RIPK3 siRNA or scrambled siRNA Fig SCD: N
• Dillon CP, Oberst A, Weinlich R et al. Survival function of the FADD-CASPASE-8-cFLIP(L) complex. Cell Rep. 2012 May 31 [PMID: 22675671]
• Oberst A, Dillon CP, Weinlich R et al. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis. Nature. 2011 Mar 17 [PMID: 21368763]
• Kim JY, Kim YJ, Lee S, Park JH. BNip3 is a mediator of TNF-induced necrotic cell death. Apoptosis. 2011 Feb [PMID: 20963496] (WB)
  
  Details:
  RIP3 (IMG-5523-1 & 5523-2). WB: A549 cells treated or untreated with TNF-alpha, Fig 4D.
• Kaiser WJ, Upton JW, Mocarski ES. Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNA-dependent activator of IFN regulatory factors. J Immunol. 2008 Nov 1 [PMID: 18941233] (WB, Human, Mouse)
  
  Details:
  WB [human 293T cells (2), mouse L929 cells (4)], Figs. 2A-C; 4B.

Review for RIPK3/RIP3 Antibody (NBP1-77299) (1)

Average Rating: 5

(Based on 1 review)

We have 1 review tested in 1 species: L929 cells.

reviewed by: Anonymous WB L929 cells 05/04/2020

Summary

• Application: Western Blot
• Sample Tested: 20 ug whole cell lysate
• Species: L929 cells
• Lot: 8337-1701

Comments

• Comments: L929 cell line
  Primary antibody: 1:2000,
  Secondary antibody: 1:20000

FAQs for RIPK3/RIP3 Antibody (NBP1-77299). (Showing 1 - 5 of 5 FAQs).

1. What research areas can this product be used in?
   • All RIPK3/RIP3 products can be used in: Cancer, Cell Biology, Immunology, Necroptosis, Protein Kinase, Signal Transduction.
2. What is the theoretical molecular weight for your RIPK3/RIP3 antibodies?
   • This depends on the immunogen. We have one RIPK3/RIP3 antibody [NBP1-77299] that is raised against murine RIP3. The theoretical molecular weight for this product is 53 kDa. The rest of our RIPK3/RIP3 antibodies are developed against human RIP3 and have a theoretical molecular weight of 56.7 kDa.
3. If this product is used in an application or species as a part of a customer review, will that validate this product in the application/species?
   • If any of our primary antibodes are used in an untested application or species and it is shown to work through images from customer reviews or through publications, this validates the application/species for this product. Please check out our Innovator's Reward Program if you decide to test an antibody with a species or application that is not currently listed.
4. I am currently looking for a good antibody working as an ELISA capture antibody for mouse RIP3. Would you let me know if this application was tested during the development of your NovusBio (NBP1-77299) antibody.
   • Our product NBP1-77299 has been validated for use in ELISA. We tested this product in a direct ELISA format; it has not yet been tested in a sandwich ELISA format. The immunogen used to make this product falls between amino acids 473 to 486 of the mouse protein. If your detection antibody is outside of this range, it is possible that this combination will yield positive results.
5. What human isoforms can be detected by this antibody?
   • RIPK3/RIP3 Antibody [NBP1-77299] can detect human isoform 1. This antibody can also detect isoforms of mouse and rat.

Bioinformatics

• Gene Symbol: RIPK3
• Entrez:
  • 6063101(Mouse)
• Uniprot:
  • AAF03133(Mouse)