RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

Novus Biologicals, part of Bio-Techne | Catalog # NBP3-32795

Recombinant Monoclonal Antibody
Novus Biologicals, part of Bio-Techne
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NBP3-32795
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Key Product Details

Species Reactivity

Mouse, Rat

Applications

Immunocytochemistry/ Immunofluorescence, Western Blot

Label

Unconjugated

Antibody Source

Recombinant Monoclonal Rabbit IgG Clone # JE44-62

Concentration

1 mg/ml

View all RIPK3/RIP3 Antibodies »

Product Specifications

Immunogen

Synthetic phospho-peptide corresponding to residues surrounding Ser232 of Human RIPK3/RIP3. (Uniprot: Q9QZL0)

Modification

p Ser 232

Localization

Cytoplasm, cytosol, Nucleus.

Clonality

Monoclonal

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.

Scientific Data Images for RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

Western Blot: RIPK3/RIP3 [p Ser232] Antibody (JE44-62) [NBP3-32795]

Western Blot: RIPK3/RIP3 [p Ser232] Antibody (JE44-62) [NBP3-32795] - Western blot analysis of RIPK3/RIP3 on different lysates with Rabbit anti-RIPK3/RIP3 antibody (NBP3-32795) at 1/1,000 dilution.

Lane 1: L-929 whole cell lysate
Lane 2: L-929 treated with 20uM Z-VAD for 3.5 hours, add 100nM SM-164 and 20ng/ml TNF-a for 3 hours whole cell lysate
Lane 3: L-929 treated with pp for 1 hour whole cell lysate
Lane 4: L-929 treated with 20uM Z-VAD for 3.5 hours, add 100nM SM-164 and 20ng/ml TNF-a for 3 hours, treated with pp for 1 hour whole cell lysate

Lysates/proteins at 20 ug/Lane.

Predicted band size: 53 kDa
Observed band size: 53 kDa

Exposure time: 20 seconds;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (NBP3-32795) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody at 1:100,000 dilution was used for 1 hour at room temperature.
RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

Immunocytochemistry/ Immunofluorescence: RIPK3/RIP3 [p Ser232] Antibody (JE44-62) [NBP3-32795]

Immunocytochemistry/ Immunofluorescence: RIPK3/RIP3 [p Ser232] Antibody (JE44-62) [NBP3-32795] - Immunocytochemistry analysis of L-929 cells treated with or without 20μM Z-VAD for 2.75 hours, add 100nM SM-164 and 20ng/ml TNF-alpha for 2.25 hours, then treated with lambdapp for 1 hour labeling RIPK3/RIP3 with Rabbit anti-RIPK3/RIP3 antibody (NBP3-32795) at 1/100 dilution.

Cells were fixed in 4% paraformaldehyde for 10 minutes at 37 ℃, permeabilized with 0.05% Triton X-100 in PBS for 20 minutes, and then blocked with 2% negative goat serum for 30 minutes at room temperature. Cells were then incubated with Rabbit anti-RIPK3/RIP3 antibody (NBP3-32795) at 1/100 dilution in 2% negative goat serum overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488) was used as the secondary antibody at 1/1,000 dilution. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594) was used as the secondary antibody at 1/1,000 dilution.

Applications for RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

Application
Recommended Usage

Immunocytochemistry/ Immunofluorescence

1:100

Western Blot

1:1000

Formulation, Preparation, and Storage

Purification

Protein A purified

Formulation

1*TBS (pH7.4), 0.05% BSA and 40% Glycerol

Preservative

0.05% 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 short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

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

Gene Symbol

RIPK3

Additional RIPK3/RIP3 Products

Product Documents for RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

Product Datasheets

Certificate of Analysis

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Product Specific Notices for RIPK3/RIP3 [p Ser232] Antibody (JE44-62)

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.

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