RIPK2 Antibody

What is RIPK2 and why is it important as a research target?

RIPK2 (Receptor-interacting serine/threonine-protein kinase 2) is a 540 amino acid kinase that plays an essential role in modulating both innate and adaptive immune responses. As an adapter protein, it functions as a critical downstream signaling molecule for Nucleotide-binding-oligomerization-domain-containing proteins 1 and 2 (NOD1 and NOD2) that recognize bacterial peptidoglycans . Upon NOD1/2 activation, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) . The significance of RIPK2 extends beyond immune regulation to potential roles in inflammatory diseases and cancer, making it an important target for antibody-based research applications.

Which cell types and tissues typically express RIPK2?

RIPK2 exhibits broad tissue distribution, being detected in heart, brain, placenta, lung, peripheral blood leukocytes, spleen, kidney, testis, prostate, pancreas, and lymph nodes . At the cellular level, RIPK2 is highly expressed in immune cells including macrophages and microglial cells, where it mediates inflammatory responses to bacterial components such as muramyl dipeptide (MDP) . When selecting appropriate positive controls for RIPK2 antibody validation, cell lines such as Ramos (human Burkitt's lymphoma), HeLa (human cervical epithelial carcinoma), HEK-293T, and THP-1 cells have been demonstrated to express detectable levels of RIPK2 protein .

What is the expected molecular weight of RIPK2 in Western blot applications?

• Approximately 62 kDa band in Ramos and HeLa cell lines under reducing conditions

• Observed molecular weight ranges of 46-55 kDa and 61-72 kDa reported in various cell types

These variations may reflect post-translational modifications, particularly ubiquitination at multiple sites (K209, K410, and K538) , or potential isoforms. When troubleshooting Western blot applications, researchers should consider that the RIPK2 band pattern might change upon activation, with an "upshift" sometimes observed following bacterial infection or pathway stimulation .

How should RIPK2 antibodies be stored to maintain optimal performance?

For optimal antibody performance and longevity:

• Store RIPK2 antibodies at -20°C to -70°C for long-term storage (12 months from receipt)

• For reconstituted antibodies, maintain at 2-8°C under sterile conditions for short-term use (up to 1 month)

• For extended storage after reconstitution, aliquot and store at -20°C to -70°C (stable for up to 6 months)

• Use manual defrost freezers and avoid repeated freeze-thaw cycles which can compromise antibody integrity

• Some formulations include stabilizers such as PBS with 0.02% sodium azide and 50% glycerol (pH 7.3) to enhance stability

The preparation of small aliquots is generally unnecessary for -20°C storage of antibodies in protective buffer formulations containing glycerol .

What are the optimal conditions for detecting RIPK2 by Western blotting?

For successful Western blot detection of RIPK2:

Sample Preparation:

• Prepare cell lysates from appropriate sources (Ramos, HeLa, THP-1, or HEK-293T cells)

• Use PVDF membrane for protein transfer

• Run samples under reducing conditions

Primary Antibody Protocol:

• Recommended dilutions range from 1:1000 to 1:8000 depending on the specific antibody

• For R&D Systems MAB103872, use 2 μg/mL concentration

• Incubate with appropriate blocking buffer (Western Blot Buffer Group 1 has been validated)

Detection:

• Use HRP-conjugated secondary antibody corresponding to the primary antibody host species

• Visualize using enhanced chemiluminescence

• Expected band size approximately 62 kDa, though multiple bands may appear due to post-translational modifications

When analyzing results, be aware that RIPK2 often displays an "upshift" pattern upon activation, which can serve as an indicator of its functional status within signaling pathways .

How can I distinguish between active and inactive RIPK2 in my experiments?

Several approaches can be employed to assess RIPK2 activation status:

• Phospho-specific antibodies: Use antibodies targeting phosphorylation sites, particularly tyrosine-474, which is an autophosphorylation site indicative of active RIPK2 .

• Mobility shift analysis: Active RIPK2 often displays an upshift in molecular weight on Western blots due to post-translational modifications, particularly ubiquitination .

• Kinase activity assays:

  • The Transcreener RIPK2 Assay measures ADP formed by enzymatic activity

  • ADP-Glo assay quantifies ADP formed from a kinase reaction

  • Radiometric in vitro kinase assay monitors the addition of 32P-γ-ATP to RIPK2

• Ubiquitination status: Active RIPK2 undergoes extensive K63-, K27- and M1-linked ubiquitination mediated by XIAP .

For research specifically focused on inflammatory pathways, activation can be confirmed by measuring downstream effects, such as phosphorylation of p65 NF-κB and p38 MAPK, which are completely blocked when RIPK2 is inhibited .

What stimuli can be used to activate RIPK2 in experimental settings?

To effectively activate RIPK2 in experimental models:

• Muramyl dipeptide (MDP): The primary agonist of NOD2 that robustly activates RIPK2-dependent signaling. In microglia, MDP has been shown to increase pro-inflammatory gene expression of Nos2, Il-1β, Tnfα, Il6, and Mmp9 in a concentration-dependent manner .

• Bacterial infection: Direct infection with bacteria containing peptidoglycan structures recognized by NOD1/2 receptors can activate RIPK2 pathways .

• NOD1 agonists: While less represented in the search results, NOD1-specific activators can also engage RIPK2-dependent pathways.

• Recombinant proteins: Purified recombinant RIPK2 can be used in in vitro kinase assays to study intrinsic activity .

It's important to note that lipopolysaccharide (LPS), while activating inflammatory pathways, primarily signals through TLR4 and appears to activate inflammatory responses largely independent of RIPK2 in microglial cells .

How can I validate the specificity of a RIPK2 antibody?

Multi-parameter validation approaches include:

• Positive and negative controls:

  • Use cell lines with known RIPK2 expression (Ramos, HeLa, THP-1) as positive controls

  • Include RIPK2 knockout or knockdown samples as negative controls

• Molecular weight verification:

  • Confirm the detection of bands at the expected molecular weight range (approximately 61-62 kDa)

  • Be aware of potential post-translational modifications that may alter migration patterns

• Orthogonal detection methods:

  • Compare results across multiple techniques (WB, IP, IF/ICC)

  • Verify with multiple antibodies targeting different epitopes of RIPK2

• Functional validation:

  • Confirm that antibody detection correlates with expected biological responses

  • Use RIPK2 degrader (PROTAC molecule GSK3728857A) to demonstrate specificity

• Sequential immunoprecipitation:

  • Perform IP with one RIPK2 antibody followed by Western blot with another targeting a different epitope

The gold standard for antibody validation includes testing in samples where the target protein has been genetically depleted through CRISPR/Cas9 or siRNA approaches .

How can RIPK2 antibodies be used to investigate NOD1/2 versus TLR signaling pathways?

RIPK2 antibodies serve as valuable tools for dissecting the relative contributions of NOD1/2 and TLR signaling pathways in inflammatory responses:

• Differential pathway analysis:

  • Use RIPK2 antibodies in combination with pathway-specific stimuli: MDP for NOD2 and LPS for TLR4

  • Monitor RIPK2 post-translational modifications following each stimulus type

  • Compare patterns of RIPK2-dependent gene expression using qPCR or RNA-seq

• Protein interaction studies:

  • Use co-immunoprecipitation with RIPK2 antibodies to identify differential binding partners in NOD1/2 versus TLR pathways

  • Combine with mass spectrometry for unbiased interactome analysis

• Phosphorylation status:

  • Employ phospho-specific antibodies to monitor differential activation patterns

  • Compare RIPK2 phosphorylation kinetics between NOD and TLR stimulation

Research has revealed that RIPK2 plays a crucial role in NOD2-mediated signaling in response to MDP, but its contribution to TLR4-mediated responses to LPS appears limited . When designing experiments to address this controversy, researchers should use highly purified ligands to avoid cross-contamination issues that may confound interpretation .

What role does RIPK2 play in ubiquitination-dependent signaling and how can this be studied?

RIPK2 undergoes extensive ubiquitination as a critical regulatory mechanism:

• XIAP-dependent ubiquitination:

  • XIAP is the essential E3 ligase for RIPK2, interacting through its BIR2 domain

  • XIAP mediates K63-linked ubiquitination at K410 and K538, which is important for NOD2 signaling

  • XIAP binding to RIPK2 recruits the linear ubiquitin chain assembly complex (LUBAC)

• Experimental approaches:

  • Immunoprecipitate RIPK2 using specific antibodies followed by Western blotting with anti-ubiquitin antibodies

  • Use linkage-specific ubiquitin antibodies to distinguish K63-, K27-, and M1-linked chains

  • Manipulate XIAP levels using siRNA knockdown or SMAC overexpression to study impact on RIPK2 ubiquitination

  • Monitor RIPK2 localization to detergent-insoluble cytosolic complexes upon activation

• Functional consequences:

  • Correlation between ubiquitination status and downstream signaling events

  • Impact on protein-protein interactions and complex formation

  • Effects on RIPK2 kinase activity and substrate specificity

Understanding the ubiquitination dynamics of RIPK2 is essential for developing targeted therapeutics that may modulate inflammatory responses in diseases associated with NOD1/2 signaling dysregulation .

How are RIPK2 antibodies used in cancer research, particularly in inflammatory breast cancer studies?

RIPK2 antibodies serve critical functions in cancer research:

• Activation status assessment:

  • Use of phospho-specific antibodies (targeting Y474) to detect activated RIPK2 in cancer tissues

  • IHC analysis reveals robust and diffuse positive cytoplasmic staining in invasive carcinomas versus non-neoplastic breast tissue

• Correlation with disease progression:

  • RIPK2 activity has been shown to correlate with advanced tumor status, metastasis, staging, and body mass index (BMI) in inflammatory breast cancer

  • IBC cell lines show significantly increased levels of RIPK2 activity compared to non-IBC cell lines like MCF10A and MCF7

• Methodology for activity measurement:

  • Western blotting with phospho-specific antibodies

  • RIPK2 ADP-Glo assay measuring ADP formed from kinase reactions

  • Radiometric in vitro kinase assays monitoring 32P-γ-ATP incorporation

• Potential as diagnostic biomarker:

  • Immunohistochemical detection of activated RIPK2 in patient samples

  • Correlation with clinical parameters and outcomes

These applications suggest that RIPK2 may serve as both a biomarker and potential therapeutic target in inflammatory forms of cancer .

What methods can be used to inhibit RIPK2 function in experimental models?

Several approaches for RIPK2 inhibition include:

• PROTAC-mediated degradation:

  • GSK3728857A PROTAC molecule induces dramatic, concentration- and time-dependent degradation of RIPK2 protein

  • Complete abolishment of MDP-induced inflammatory responses demonstrates efficacy

• Kinase inhibitors:

  • Small molecule inhibitors identified through pharmacophore modeling approaches

  • Specificity assessment using ROC curves and AUC analysis

• Genetic approaches:

  • siRNA-mediated knockdown of RIPK2

  • CRISPR/Cas9-mediated gene knockout

  • XIAP knockdown to disrupt RIPK2 ubiquitination and function

• Disruption of protein-protein interactions:

  • SMAC mimetics/SMAC overexpression to antagonize XIAP function and thus indirectly inhibit RIPK2 signaling

  • Peptides or small molecules targeting the RIPK2-NOD2 interaction

When selecting inhibition strategies, researchers should consider that some RIPK2 inhibitors may have dual mechanisms - directly inhibiting kinase activity while also blocking the interaction with XIAP , which could affect experimental interpretation.

Why might I observe multiple bands when using RIPK2 antibodies in Western blot applications?

Multiple bands in RIPK2 Western blots may result from:

• Post-translational modifications:

  • Ubiquitination at multiple sites (K209, K410, K538) can cause significant molecular weight shifts

  • Phosphorylation events, particularly autophosphorylation at sites like Y474

  • Active RIPK2 often displays an "upshift" pattern following pathway activation

• Protein isoforms:

  • Alternative splice variants

  • Proteolytic processing

• Experimental parameters:

  • Incomplete denaturation of protein complexes

  • Protein degradation during sample preparation

  • Cross-reactivity with related proteins in the RIP kinase family

Comprehensive approach to resolving multiple band issues:

• Compare patterns across multiple antibodies targeting different epitopes

• Include positive controls with known RIPK2 expression

• Perform phosphatase or deubiquitinase treatment of lysates

• Use knockout or knockdown samples to confirm specificity

Observed molecular weight ranges of 46-55 kDa and 61-72 kDa have been reported in literature , with the expected molecular weight of unmodified RIPK2 being approximately 61 kDa.

How should I interpret conflicting results between RIPK2 expression and activation patterns?

When facing discrepancies in RIPK2 data:

• Distinguish between expression and activation:

  • RIPK2 protein levels (detected by total RIPK2 antibodies) may not correlate with activity

  • Phosphorylation and ubiquitination status (detected by modification-specific antibodies) reflect activation

  • Kinase activity assays measure functional output independent of modifications

• Consider cell type and context specificities:

  • RIPK2 signaling dynamics differ between cell types (e.g., microglial cells vs. macrophages)

  • Stimulus-specific responses (MDP vs. LPS) may vary within the same cell type

  • Presence of regulatory proteins like XIAP impacts RIPK2 function

• Methodological reconciliation:

  • Compare protein levels (Western blot) with kinase activity (ADP-Glo, Transcreener assays)

  • Assess downstream signaling events (NF-κB activation, cytokine production)

  • Examine RIPK2 localization changes using fractionation or microscopy

• Temporal considerations:

  • RIPK2 activation is dynamic with specific kinetics

  • XIAP levels may decline upon bacterial infection, coinciding with RIPK2 modification

When publishing potentially conflicting results, clearly document experimental conditions, stimuli concentrations, timing, and cell types to facilitate accurate interpretation and reproducibility.

What controls should be included when using RIPK2 antibodies for immunohistochemistry in tissue samples?

For rigorous immunohistochemical detection of RIPK2:

• Positive tissue controls:

  • Include tissues with known RIPK2 expression (lymph nodes, spleen, peripheral blood leukocytes)

  • For cancer studies, inflammatory breast cancer samples have shown robust RIPK2 staining

• Negative tissue controls:

  • Non-neoplastic breast tissue has been used as a control in cancer studies

  • Tissues known to express minimal RIPK2

• Technical controls:

  • Omission of primary antibody

  • Isotype control antibody at matching concentration

  • Concentration gradients to determine optimal antibody dilution

  • Antigen retrieval optimization

• Validation controls:

  • Peptide competition assays to confirm specificity

  • Correlation with Western blot data from the same samples

  • Comparison of staining with multiple antibodies targeting different epitopes

• Biological interpretation controls:

  • Correlation of staining pattern with expected subcellular localization (primarily cytoplasmic)

  • Comparison between inactive and stimulated tissues/cells

For accurate scoring and interpretation, develop a standardized evaluation system considering staining intensity, percentage of positive cells, and subcellular localization patterns.

How can RIPK2 antibodies be applied in studying inflammatory neurological disorders?

RIPK2 antibodies offer valuable insights in neuroinflammatory research:

• Microglial activation studies:

  • RIPK2 plays a crucial role in microglial inflammatory responses to bacterial muramyl dipeptide

  • Antibodies can track RIPK2 activation status in microglia from various neurological disease models

• Pathway-specific neuroinflammation:

  • Distinguish between NOD-dependent and TLR-dependent inflammatory responses in CNS

  • Investigate differential roles in acute versus chronic neuroinflammation

• Experimental approaches:

  • Immunohistochemical detection in brain tissue from neurological disease models

  • Flow cytometry of isolated microglia to quantify RIPK2 activation

  • Western blot analysis of brain region-specific RIPK2 signaling

• Therapeutic target identification:

  • Correlation between RIPK2 activation and disease progression

  • Testing RIPK2 inhibitors or PROTACs in neuroinflammatory models

  • Combining with behavioral assessments to link molecular changes to functional outcomes

The importance of RIPK2 in microglial NOD2 signaling suggests potential relevance to neurological diseases with infectious or inflammatory components, including multiple sclerosis, Alzheimer's disease, and neurological manifestations of inflammatory bowel disease .

What are the latest advancements in high-throughput screening methods using RIPK2 antibodies?

Recent developments in high-throughput RIPK2 research include:

• Transcreener RIPK2 Assay:

  • Directly measures ADP formed by RIPK2 enzymatic activity

  • Available with multiple detection modalities: FP, FI, and TR-FRET

  • Compatible with 96, 384, and 1536-well formats

  • Simple mix-and-read format amenable to high-throughput screening

• Assay performance metrics:

  • Z' factor measurements using optimized RIPK2 reaction conditions demonstrate robust assay performance (Z' = 0.74)

  • Linearity when raw data is converted to ADP using a standard curve

  • Standardized conditions: 50 μM ATP, 40 mM Tris (pH 7.5), 2.5 mM MnCl2, and 20 mM MgCl2

• PROTAC screening applications:

  • GSK3728857A PROTAC validation demonstrates concentration- and time-dependent RIPK2 degradation

  • Potential framework for screening novel PROTAC molecules targeting RIPK2

• Inhibitor screening platforms:

  • Pharmacophore models with high specificity and sensitivity to detect/select RIPK2 inhibitors

  • ROC curve analysis and AUC evaluation for screening optimization

  • AUC > 0.70 considered to have moderate predictive ability for RIPK2 inhibitor identification

These technologies facilitate the discovery of new modulators of RIPK2 activity with potential therapeutic applications in inflammatory and immune-related disorders.

How can phospho-specific RIPK2 antibodies enhance our understanding of signaling dynamics?

Phospho-specific RIPK2 antibodies provide crucial insights into activation mechanisms:

• Key phosphorylation sites:

  • Tyrosine-474 is a critical autophosphorylation site indicative of active RIPK2

  • Phospho-specific antibodies targeting this site enable specific detection of activated RIPK2

• Temporal signaling analysis:

  • Time-course experiments using phospho-antibodies reveal activation kinetics

  • Correlation between phosphorylation patterns and downstream signaling events

• Advanced applications:

  • Intracellular flow cytometry for single-cell analysis of RIPK2 activation

  • Multiplexed imaging to correlate RIPK2 phosphorylation with other pathway components

  • Phosphoproteomics integration for comprehensive signaling network analysis

• Clinical correlations:

  • Phospho-RIPK2 as a biomarker in inflammatory diseases and cancer

  • IHC analysis of phospho-RIPK2 in patient samples correlates with advanced tumor status and metastasis

Phospho-specific antibodies have demonstrated utility in distinguishing inflammatory breast cancer from non-IBC cell lines and tissues, suggesting potential diagnostic applications beyond basic research .

What are the critical parameters for immunoprecipitation of RIPK2?

For successful RIPK2 immunoprecipitation:

• Antibody selection and amount:

  • Use 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

  • Select antibodies validated specifically for IP applications

• Lysis conditions:

  • Optimize buffer composition to preserve protein-protein interactions

  • Consider detergent selection carefully, as RIPK2 can localize to detergent-insoluble cytosolic complexes upon activation

• Co-immunoprecipitation considerations:

  • Gentler lysis conditions may be required to maintain native complex interactions

  • Cross-linking may help capture transient interactions

• Controls:

  • Include isotype control antibodies

  • Use RIPK2-deficient cells as negative controls

  • Include input samples for quantitative assessment

• Detection strategy:

  • Western blot with a different RIPK2 antibody recognizing a distinct epitope

  • Mass spectrometry for unbiased identification of interacting partners

For studying ubiquitination, include deubiquitinase inhibitors in lysis buffers. When investigating phosphorylation, incorporate phosphatase inhibitors to preserve modification status during sample preparation.

How should researchers accurately quantify RIPK2 expression and activity levels?

Multiple complementary approaches ensure accurate RIPK2 quantification:

• Protein expression quantification:

  • Western blot with housekeeping protein normalization

  • ELISA for absolute quantification

  • Mass spectrometry for label-free or labeled quantification

• Activity assessment:

  • ADP-Glo assay that measures ADP formed from kinase reactions

  • Transcreener RIPK2 Assay with standard curve conversion for linearity

  • Radiometric in vitro kinase assay monitoring 32P-γ-ATP incorporation

  • Densitometric analysis of phospho-specific Western blots

• Standardization considerations:

  • Include recombinant RIPK2 standards

  • Validate antibody linear range

  • Use multiple cell lines with known RIPK2 expression levels as references

• Functional readouts:

  • Downstream phosphorylation events (p65 NF-κB, p38 MAPK)

  • Inflammatory gene expression (Nos2, Il-1β, Tnfα, Il6, etc.)

  • Cytokine secretion measurements

When publishing quantitative RIPK2 data, clearly report normalization methods, antibody dilutions, exposure parameters, and calculation approaches to ensure reproducibility across research groups.

What approaches resolve inconsistencies between different RIPK2 antibodies in research applications?

When facing antibody inconsistencies:

• Epitope mapping and comparison:

  • Identify the specific regions recognized by each antibody

  • Assess potential epitope masking by protein interactions or modifications

  • Consider accessibility of epitopes in different experimental conditions

• Validation using multiple detection methods:

  • Compare results across techniques (WB, IF, IP, IHC)

  • Verify with orthogonal non-antibody based methods (mass spectrometry)

  • Correlate with functional readouts of RIPK2 activity

• Systematic troubleshooting:

  • Test different sample preparation methods

  • Optimize blocking conditions and antibody dilutions

  • Compare different secondary antibody detection systems

• Definitive validation:

  • Test all antibodies in RIPK2 knockout/knockdown samples

  • Use peptide competition assays

  • Evaluate antibody performance in overexpression systems

• Reporting and transparency:

  • Document batch numbers and full antibody details

  • Report all optimization steps and conditions

  • Share negative or inconsistent results to advance field knowledge

When conducting multi-antibody comparisons, create a systematic validation matrix documenting performance across applications, cell types, and conditions to guide optimal antibody selection for specific research questions.