Key Product Details

Validated by

Knockout/Knockdown

Species Reactivity

Validated:

Human, Mouse, Rat

Cited:

Human, Mouse, Rat

Applications

Validated:

Knockout Validated, Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, ELISA, Immunocytochemistry/ Immunofluorescence, Simple Western, Immunoprecipitation, Gel Supershift Assay, Knockdown Validated

Cited:

Knockout Validated, Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Immunocytochemistry/ Immunofluorescence, Immunoprecipitation, Proximity Ligation Assay, Gel Supershift Assay, IF/IHC, Knockdown Validated

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Format

BSA Free
View all RIPK3/RIP3 Primary Antibodies »

Product Specifications

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

Reactivity Notes

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

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.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

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.

Description

Novus Biologicals Knockout (KO) Validated Rabbit RIPK3/RIP3 Antibody - BSA Free (NBP1-77299) is a polyclonal antibody validated for use in IHC, WB, ELISA, ICC/IF, Simple Western and IP. Anti-RIPK3/RIP3 Antibody: Cited in 81 publications. All Novus Biologicals antibodies are covered by our 100% guarantee.

Scientific Data Images for RIPK3/RIP3 Antibody - BSA Free

Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

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 AntibodyBSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - Mouse Cell lines. 15 ug of lysates per lane. Antibodies: [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.
Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

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.
Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 AntibodyBSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 Antibody [NBP1-77299] - 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.
Knockout Validated: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299]

Western Blot: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299]

RIPK3-RIP3-Antibody-Knockout-Validated-NBP1-77299-img0025.jpg
Knockdown Validated: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299]

Knockdown Validated: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299]

RIPK3-RIP3-Antibody-Knockdown-Validated-NBP1-77299-img0026.jpg
RIPK3/RIP3 Antibody - BSA Free

Western Blot: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] -

Western Blot: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] - Bone-marrow-derived macrophages exhibit significant reduction in RIPK3 levels in Ripk3 delta Global, Ripk3 delta MΦ-Cre & Ripk3 delta EC-Cre mice. (A,B) BMDMs were isolated, differentiated on chamber slides for 7 days, immunostained for macrophage markers CD68 (red; A) & CD11b (pink; B), & co-stained for nuclei (Hoechst; blue). All cells from seven separate BMDM isolations were positive for these markers (n=7). (C) A phagocytosis assay was performed on BMDMs with fluorescent polystyrene beads. Phagocytic cells display TRITC+/Hoechst+ signal, & 85% of cells from two separate BMDM isolations were double positive (n=2). (D-F) Protein or RNA was collected from control, Ripk3 delta Global, Ripk3 delta MΦ-Cre & Ripk3 delta EC-Cre BMDMs. RNA was converted to cDNA & analyzed by qPCR for Ripk3 levels (D). Protein lysates were immunoblotted to identify CD11b, RIPK3 & beta -actin (loading control) (E) & quantified (F). (G) RNA from control & Ripk3 delta Global BMDMs was converted to cDNA & analyzed by qPCR for Ripk3-Exon 10 levels. For panels D,F,G, each dot represents a BMDM isolation from an individual animal. Statistics for panels D & F were calculated using one-way ANOVA with Dunnett's multiple comparisons test. Overall ANOVA P-values (prior to the post hoc tests) are 0.0002 (D) & <0.0001 (F). Statistics for panel G were calculated using an unpaired t-test with Welch's correction. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Data are mean±s.d. Scale bars: 25 µm. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31953345), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
RIPK3/RIP3 Antibody - BSA Free

Western Blot: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] -

Western Blot: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] - p-MLKL levels are nearly undetectable in advanced plaques. (A-C) After 3 months on a Western diet, protein was collected from control (n=7), Ripk3 delta MΦ-Cre (n=5), Ripk3 delta EC-Cre (n=3) & Ripk3 delta SMC-Cre (n=3) aortas. Protein lysates were immunoblotted to identify p-MLKL (Abcam; #ab196436), MLKL & beta -actin (loading control) (A) & quantified (B,C). A faint positive p-MLKL signal can be seen in the representative blot in the control arch region; however, we could only detect p-MLKL in two out of the 18 aortas analyzed (B). Note that the same transfer membrane used for detecting CD11b (in Fig. 4S) was reprobed for p-MLKL & MLKL in A; the beta -actin control blots are therefore the same in both figures. (D,E) Protein lysates were immunoblotted to identify RIPK3 & GAPDH (loading control) (D) & quantified (E). For panels B,C,E, each dot represents an individual animal. Statistics were calculated using two-way ANOVA. Overall ANOVA P-values are 0.76 (B), 0.07 (C) & 0.46 (E). Data are mean±s.d. Image collected & cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/31953345), licensed under a CC-BY license. Not internally tested by Novus Biologicals.
RIPK3/RIP3 Antibody - BSA Free

Immunocytochemistry/ Immunofluorescence: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] -

Immunocytochemistry/ Immunofluorescence: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] - Validation of RIPK3/RIP3 in Rat Kidney Tissue. Immunofluorescent analysis of 4% paraformaldehyde-fixed Rat Kidney tissue labeling RIPK3/RIP3 with at 20 ug/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).
RIPK3/RIP3 Antibody - BSA Free

Immunohistochemistry: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] -

Immunohistochemistry: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] - Validation of RIPK3/RIP3 in Mouse Kidney Tissue. Immunohistochemical analysis of paraffin-embedded mouse kidney tissue using anti-RIPK3/RIP3 antibody at 2.5 ug/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
RIPK3/RIP3 Antibody - BSA Free

Immunohistochemistry: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] -

Immunohistochemistry: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] - Validation of RIPK3/RIP3 in Rat Kidney Tissue.Immunohistochemical analysis of paraffin-embedded rat kidney tissue using anti-RIPK3/RIP3 antibody at 5 ug/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.
RIPK3/RIP3 Antibody - BSA Free

Western Blot: RIPK3/RIP3 Antibody - BSA Free [NBP1-77299] -

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

Applications for RIPK3/RIP3 Antibody - BSA Free

Application
Recommended Usage

ELISA

1:100 - 1:2000

Immunocytochemistry/ Immunofluorescence

20 ug/ml

Immunohistochemistry

5 ug/ml

Immunohistochemistry-Paraffin

5 ug/ml

Immunoprecipitation

20 ug/mL

Simple Western

1:25

Western Blot

0.1-0.5 ug/ml
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, IHC, and WB reported in multiple pieces of scientific literature.
See Simple Western Antibody Database for Simple Western validation: Tested in Mouse, rat heart tissue 0.25 mg/mL, separated by Size, antibody dilution of 1:25, apparent MW was 70 kDa

Reviewed Applications

Read 1 review rated 5 using NBP1-77299 in the following applications:

Formulation, Preparation, and Storage

Purification

Peptide affinity purified

Formulation

PBS

Format

BSA Free

Preservative

0.02% Sodium Azide

Concentration

1 mg/ml

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Store at 4C.

Background: RIPK3/RIP3

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

Long Name

Receptor (TNFRSF)-Interacting Serine-Threonine Kinase 3

Alternate Names

RIP3

Entrez Gene IDs

6063101 (Mouse)

Gene Symbol

RIPK3

UniProt

AAF03133 (Mouse)

Additional RIPK3/RIP3 Products

Product Documents for RIPK3/RIP3 Antibody - BSA Free

Certificate of Analysis

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Product Specific Notices for RIPK3/RIP3 Antibody - BSA Free

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.

Related Research Areas

Apoptosis Adaptor Proteins
NFkB Pathway

Citations for RIPK3/RIP3 Antibody - BSA Free

Customer Reviews for RIPK3/RIP3 Antibody - BSA Free (1)

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  • RIPK3/RIP3 Antibody
    Name: Anonymous
    Application: Western Blot
    Sample Tested: 20 ug whole cell lysate
    Species: L929 cells
    Verified Customer | Posted 05/04/2020
    L929 cell line Primary antibody: 1:2000, Secondary antibody: 1:20000
    L929 cell line Primary antibody: 1:2000, Secondary antibody: 1:20000
    RIPK3/RIP3 Antibody - BSA Free NBP1-77299

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Protocols

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FAQs for RIPK3/RIP3 Antibody - BSA Free

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    • Q: 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.
      A: 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.

    • Q: 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?
      A: 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.

    • A: RIPK3/RIP3 Antibody [NBP1-77299] can detect human isoform 1. This antibody can also detect isoforms of mouse and rat.

    • A: 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.

    • A: All RIPK3/RIP3 products can be used in: Cancer, Cell Biology, Immunology, Necroptosis, Protein Kinase, Signal Transduction.
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