RIPK1 Antibody, HRP conjugated

What is RIPK1 and why is it a significant target for antibody-based detection?

RIPK1 (Receptor-interacting serine/threonine-protein kinase 1) is a key regulatory protein (approximately 75.9 kDa) that functions at the intersection of multiple cell death and inflammatory pathways. It exhibits dual functionality: kinase-dependent roles that regulate cell death and kinase-independent scaffold functions that regulate inflammatory signaling and cell survival . As a central component in TNF receptor signaling, RIPK1 participates in three critical cellular processes:

• NF-κB activation (pro-survival)

• Apoptosis regulation through complex IIa (RIPK1-FADD-CASP8)

• Necroptosis modulation via complex IIb (RIPK1-RIPK3-MLKL)

This multifunctional nature makes RIPK1 detection crucial for understanding cell fate decisions in inflammatory and immune responses, making RIPK1 antibodies essential research tools.

How do HRP-conjugated RIPK1 antibodies differ from unconjugated versions in research applications?

HRP-conjugated RIPK1 antibodies provide direct detection capability without requiring secondary antibodies, offering several methodological advantages:

When using HRP-conjugated antibodies, researchers should optimize concentrations (typically 0.5-1.0 μg/mL for Western blotting) and include proper controls to ensure specific detection of RIPK1 .

What experimental validation techniques confirm RIPK1 antibody specificity?

Validation of RIPK1 antibody specificity requires multiple complementary approaches:

• Knockout cell line validation: Compare detection between parental and RIPK1 knockout cell lines (e.g., MCF-7 human breast cancer cell line). The absence of signal in knockout lines confirms specificity .

• Multiple cell line testing: Verify consistent detection across species and cell types. For example, RIPK1 detection at approximately 75 kDa in Raji, Jurkat, DA3 (mouse), and L6 (rat) cell lines demonstrates cross-species specificity .

• Phosphorylation-specific validation: For phospho-specific antibodies (e.g., phospho-S166), treatment with kinase inhibitors like necrostatin-1 should reduce detection .

• Immunohistochemical comparison: Compare staining patterns between wild-type and knockout tissue sections, quantifying signal ratio and performance index .

• Immunoprecipitation followed by mass spectrometry: Confirm antibody pulls down RIPK1 exclusively without non-specific interactions .

Comprehensive validation across these methods ensures reliable experimental outcomes when using RIPK1 antibodies.

What are the optimal protocols for using HRP-conjugated RIPK1 antibodies in Western blot analysis?

For optimal Western blot analysis with HRP-conjugated RIPK1 antibodies:

Sample preparation and electrophoresis:

• Prepare whole cell lysates (typically 30 μg protein per lane)

• Use 7.5% SDS-PAGE for better separation of high molecular weight RIPK1 (~75-78 kDa)

• Include reducing conditions with Immunoblot Buffer Group 2

Blotting and detection protocol:

• Transfer proteins to PVDF membrane (preferred over nitrocellulose)

• Block membrane with 5% non-fat milk or BSA in TBST for 1 hour

• Incubate with HRP-conjugated RIPK1 antibody at 0.5-1.0 μg/mL overnight at 4°C

• Wash 3-5 times with TBST

• Proceed directly to chemiluminescent detection without secondary antibody

Important controls:

• Positive control: Jurkat or MCF-7 cell lysates (well-characterized RIPK1 expression)

• Negative control: RIPK1 knockout MCF-7 cell line

• Loading control: GAPDH (use separately, not as a multiplex)

Note: Be aware that some RIPK1 antibodies may detect non-specific bands, particularly at 230 kDa in Simple Western analysis systems .

How should researchers optimize immunocytochemistry/immunofluorescence protocols for RIPK1 detection?

For optimal immunocytochemical detection of RIPK1:

Fixation method comparison:
Methanol fixation typically yields stronger RIPK1 signals compared to paraformaldehyde fixation, particularly for antibodies targeting the N-terminal domain (e.g., clone D94C12) or C-terminal region (e.g., clone 38/RIP) .

Protocol optimization:

• Fix cells in cold methanol (-20°C) for 10 minutes

• Permeabilize with 0.1% Triton X-100 if using paraformaldehyde fixation

• Block with 5% normal serum from the same species as secondary antibody

• Incubate with primary RIPK1 antibody (25 μg/mL for 3 hours at room temperature)

• Wash thoroughly with PBS

• Apply fluorophore-conjugated secondary antibody (e.g., NorthernLights™ 557-conjugated Anti-Mouse IgG)

• Counterstain nuclei with DAPI

• Mount and observe using confocal microscopy

Subcellular localization:
RIPK1 generally displays cytoplasmic localization in immunofluorescence staining . This pattern should be consistent across different cell types and can serve as an additional specificity control.

What are the key differences in detecting RIPK1 versus phosphorylated RIPK1 in research samples?

Detection of total versus phosphorylated RIPK1 requires distinct methodological approaches:

For phospho-RIPK1 detection, researchers should:

• Include phosphatase inhibitors in all buffers

• Use stimulation controls (e.g., TNF-α treatment increases S166 phosphorylation)

• Include inhibitor controls (e.g., necrostatin-1 should reduce phospho-S166 signal)

• Validate with phospho-null mutants where possible

Phosphorylation at different sites has distinct functional implications: S166 relates to kinase activation, while Y384/Y383 phosphorylation (by JAK1 and SRC) suppresses TNF-induced cell death .

How can researchers effectively use RIPK1 antibodies to distinguish between necroptosis and apoptosis in cell death studies?

Distinguishing RIPK1-mediated necroptosis from apoptosis requires a methodical antibody-based approach:

Antibody panel for pathway discrimination:

Experimental design for pathway identification:

• Induce cell death with appropriate stimuli (e.g., TNF-α + smac mimetic + z-VAD-fmk for necroptosis; TNF-α + smac mimetic for apoptosis)

• Collect protein at multiple time points (0, 2, 4, 8 hours)

• Perform Western blot analysis with phospho-specific antibodies

• Use proximity ligation assays to detect RIPK1-RIPK3 (necroptosis) or RIPK1-FADD-Caspase-8 (apoptosis) complexes

• Confirm with inhibitor controls: necrostatin-1 (RIPK1 inhibitor) should block both pathways, while GSK'872 (RIPK3 inhibitor) blocks only necroptosis

Important consideration: The intermediate domain of RIPK1 has anti-apoptotic functions, and RIPK1ΔID mutants shift TNF-induced necroptosis to RIPK1 kinase-dependent apoptosis . This nuance must be considered when interpreting antibody-based detection results.

What methodological approaches ensure accurate immunohistochemical detection of RIPK1 in tissue samples?

Optimization of RIPK1 immunohistochemistry requires systematic protocol development:

Antigen retrieval method comparison:
Citrate buffer (pH 6.0) for 15 minutes has been validated for optimal RIPK1 epitope exposure in paraffin-embedded tissues . Alternative methods (EDTA-based, enzymatic) should be empirically compared if signal is suboptimal.

Protocol optimization workflow:

• Test multiple antibody concentrations (1:100 to 1:1000) on control tissues

• Compare different antigen retrieval methods

• Quantify signal-to-noise ratio for each condition

• Validate specificity using knockout tissue sections

• Calculate performance index using the formula: (wild-type signal/knockout signal ratio × integrated signal intensity)

Optimized protocol for formalin-fixed paraffin-embedded tissues:

• Deparaffinize and rehydrate sections

• Perform heat-induced epitope retrieval with citrate buffer (pH 6.0) for 15 minutes

• Block endogenous peroxidase with 3% H₂O₂

• Block non-specific binding with 5% normal serum

• Incubate with RIPK1 antibody at 1:500 dilution

• Apply HRP-conjugated secondary antibody (if primary is not HRP-conjugated)

• Develop with DAB substrate

• Counterstain, dehydrate, and mount

Researchers should note that RIPK1 typically shows cytoplasmic localization in tissues and may demonstrate differential expression patterns across tissue types .

How does pH affect RIPK1 activity and antibody detection, and what methodological controls should be implemented?

pH significantly impacts RIPK1 kinase activity and possibly antibody binding:

pH-dependent RIPK1 activity modulation:
Acidification inhibits RIPK1 kinase activity and TNF-induced cell death in a reversible manner. This pH sensitivity is mediated by histidine residues, particularly His151, which function as proton acceptors . This has implications for both experimental design and interpretation of results.

Methodological considerations for pH effects:

Recommended controls and approaches:

• Monitor and standardize culture medium pH in cell-based experiments

• For high-density cultures, control for pH-dependent effects by comparing to pH-matched low-density cultures

• Include His151 mutant RIPK1 (insensitive to pH changes) as a control where possible

• Use phospho-S166 RIPK1 antibodies to monitor kinase activity across pH conditions

• For in vitro kinase assays, test activity across a pH range (6.5-7.5)

Understanding these pH effects is particularly important when studying RIPK1 in pathological conditions where intracellular pH may be altered, such as in tumors or inflammatory environments.

What approaches can researchers use to study RIPK1-TBK1 interactions and their role in type I interferon production?

Studying RIPK1-TBK1 interactions requires specialized immunological techniques:

Co-immunoprecipitation protocol optimization:

• Lyse cells in buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 5 mM EDTA, with protease and phosphatase inhibitors

• Pre-clear lysates with protein A/G beads

• Incubate with anti-RIPK1 antibody (5 μg) overnight at 4°C

• Add protein A/G beads and incubate for 2 hours

• Wash extensively with lysis buffer

• Elute complexes by boiling in SDS sample buffer

• Analyze by Western blotting for TBK1 and phospho-TBK1

Experimental design to assess RIPK1-TBK1 pathway:

Research has demonstrated that caspase-8 negatively regulates type I IFN production by inhibiting the RIPK1-TBK1 axis during homeostasis across multiple cell types . Without caspase-8, RIPK1 interacts with TBK1 more robustly, promoting TBK1 phosphorylation in a RIPK1 kinase-dependent manner to enhance type I IFN production.

Recommended stimulation conditions for pathway analysis:

• cGAS-STING pathway: c-di-GMP transfection

• RIG-I/MDA5-MAVS pathway: poly(I:C) transfection

• TLR3/4-TRIF pathway: poly(I:C) treatment

How can RIPK1 inhibition be monitored through antibody-based techniques in the context of therapeutic development?

Monitoring RIPK1 inhibition during therapeutic development requires systematic antibody-based detection approaches:

Biomarker panel for RIPK1 inhibition:

Assay for RIPK1 degrader efficacy:
Recently developed RIPK1 degraders like LD4172 can be monitored using antibody-based techniques to assess degradation efficacy and selectivity . The protocol involves:

• Treat cells with degrader compounds at various concentrations (10 nM to 10 μM)

• Harvest cells at multiple time points (2, 4, 8, 24 hours)

• Perform Western blot analysis with RIPK1 antibodies

• Quantify RIPK1 protein levels normalized to loading controls

• Calculate DC₅₀ (concentration for 50% degradation) and Dmax (maximum degradation)

When evaluating RIPK1 kinase inhibitors like ZJU-37 (which has higher potency than Nec-1s), researchers should also examine downstream effects on oligodendrocyte progenitor cell proliferation and remyelination .

What methodological approaches are needed to study tyrosine phosphorylation of RIPK1 and its regulatory functions?

Studying tyrosine phosphorylation of RIPK1 requires specialized techniques:

Experimental detection protocol:

• Immunoprecipitate RIPK1 from cell lysates using specific antibodies

• Perform Western blotting with anti-phosphotyrosine antibodies

• Confirm specific sites with phospho-specific antibodies (e.g., pY384/pY383)

• Validate with phospho-null mutants (e.g., RIPK1^Y383F/Y383F)

• Assess functional impact using kinase activity assays

In vivo validation approach:
Analysis of tissues from Ripk1^Y383F/Y383F mutant mice reveals:

• Development of systemic inflammation

• Emergency hematopoiesis

• Enhanced TNF-induced apoptosis and necroptosis

• Impaired recruitment and activation of MK2

• These phenotypes are largely alleviated by RIPK1 kinase inhibition

Kinase identification strategy:

• Treat cells with specific kinase inhibitors (JAK inhibitors, SRC inhibitors)

• Perform in vitro kinase assays with recombinant JAK1 and SRC

• Analyze phosphorylation by Western blotting

• Confirm with genetic knockdown of candidate kinases

Research has demonstrated that non-receptor tyrosine kinases JAK1 and SRC can phosphorylate RIPK1 at Y384 (Y383 in murine RIPK1), leading to suppression of TNF-induced cell death . This represents a novel regulatory mechanism distinct from the well-characterized serine/threonine phosphorylation pathways.

How can researchers effectively use RIPK1 antibodies in spatial transcriptomics and advanced immunohistochemical analyses?

Integration of RIPK1 antibody-based detection with spatial transcriptomics requires specialized methodological approaches:

Immunohistochemical atlas development protocol:

• Optimize antibody conditions across multiple parameters (21 conditions) for each target (Caspase-8, RIPK1, RIPK3, MLKL)

• Quantify signal from wild-type versus knockout tissue

• Calculate signal ratio and performance index

• Develop automated immunohistochemistry protocols for consistent detection

Spatial transcriptomics integration workflow:

• Perform immunohistochemistry for RIPK1 on tissue sections

• Process adjacent sections for spatial transcriptomics

• Analyze gene expression data using computational approaches:

  • Construct matrices from gene clusters

  • Reduce to aggregated vectors

  • Select top scores within percentile of maximum

  • Aggregate corresponding Leiden clusters hierarchically

  • Calculate log-fold changes for genes of interest in each zone

This approach allows correlation between RIPK1 protein expression patterns and transcriptional signatures across tissue microenvironments. For spleen analysis, distinct expression patterns can be identified across white pulp, red pulp, germinal centers, and marginal zones .

Data analysis recommendations:

• Use software packages including Anndata (v0.7.5), Stereopy (v0.12.0), Scanpy (v1.9.2)

• Implement dimensionality reduction techniques for visualization

• Employ hierarchical clustering to identify spatial patterns

• Correlate protein levels with transcript abundance for Ripk1 and related genes

This integrated approach provides comprehensive understanding of RIPK1 distribution and function within complex tissue architectures.