RIPK3 Antibody

What applications are suitable for RIPK3 antibodies?

RIPK3 antibodies have been validated for multiple experimental applications including:

The application should be selected based on your experimental question. For detecting protein expression levels, Western blot is most suitable, while localization studies benefit from IF or IHC approaches .

How do I determine the appropriate molecular weight for RIPK3 detection?

When performing Western blot analysis, human RIPK3 typically appears at approximately 57-70 kDa . Specific RIPK3 bands have been detected at approximately 60 kDa in multiple cell lines including:

• SK-BR-3 human breast cancer cells

• Raji human Burkitt's lymphoma cells

• K562 human chronic myelogenous leukemia cells

• HT-29 human colon adenocarcinoma cells

It's crucial to note that the theoretical calculated molecular weight is 57 kDa (518 amino acids), but post-translational modifications may result in migration differences on SDS-PAGE gels .

What positive control cell lines should I use for RIPK3 antibody validation?

Based on extensive testing, these cell lines consistently show detectable RIPK3 expression:

These cell lines provide reliable positive controls, essential for confirming antibody specificity before proceeding to experimental samples .

How can I validate RIPK3 antibody specificity for my experimental system?

Rigorous validation is essential for reliable RIPK3 detection. A comprehensive validation approach includes:

• Genetic controls: Testing in RIPK3-deficient (Ripk3-/-) cells or tissues compared to wild-type . This represents the gold standard for specificity.

• Multiple antibody comparison: Testing several anti-RIPK3 antibodies simultaneously. Research shows significant variation in specificity among commercially available antibodies .

• Molecular weight verification: Confirming band size at the expected molecular weight (57-70 kDa for human RIPK3) .

• Cross-application testing: Verifying consistent results across multiple applications (e.g., WB, IF, IHC) .

• siRNA knockdown: Demonstrating reduced signal after RIPK3 knockdown via siRNA treatment.

One study evaluated seven anti-human RIPK3 antibodies and found that several produced non-specific signals in both immunofluorescence and immunoblotting applications. For example, clone E1Z1D showed high specificity in immunoblots but generated non-specific signals in immunofluorescence .

How do I design experiments to distinguish between RIPK3-mediated necroptosis and apoptosis?

Distinguishing between these cell death pathways requires careful experimental design:

• Molecular markers analysis:

  • Necroptosis: Monitor RIPK3-MLKL interaction and MLKL phosphorylation

  • Apoptosis: Assess caspase-8, caspase-3, and PARP cleavage

• Pharmacological inhibitors:

  • Include caspase inhibitors (e.g., QVD) to block apoptosis

  • Test necrostatin-1 to inhibit RIPK1-dependent necroptosis

  • Apply MLKL inhibitors to block necroptosis execution

• Genetic manipulation:

  • Compare responses in wild-type, Mlkl-/-, and Fadd-/- cells

  • Research shows that in Mlkl-/- cells, RIPK3 dimerization induces only apoptosis

  • In Fadd-/- cells, RIPK3 dimerization triggers only necroptosis

• Morphological assessment:

  • Electron microscopy or appropriate staining methods to distinguish cellular morphology

Research demonstrates that when RIPK3 is dimerized, the cell death mode depends on the availability of downstream molecules: with FADD and caspase-8 present, apoptosis occurs; with MLKL present, necroptosis predominates .

How does RIPK3 expression vary across different tissues and what implications does this have for antibody detection?

RIPK3 expression exhibits significant tissue variability, which directly impacts detection approaches:

• Tissue expression patterns: Studies in chicken models revealed that RIPK3 protein is most abundantly expressed in the liver and kidney, with lower expression in other tissues .

• Antibody sensitivity requirements: Tissues with low RIPK3 expression require more sensitive detection methods:

  • Consider signal amplification systems for IHC

  • Use highly concentrated lysates for WB

  • Longer exposure times may be necessary for less abundant expression

• Background considerations: Higher antibody concentrations needed for low-expressing tissues may increase background signals. Sequential optimization is recommended:

  • Start with positive control tissues (e.g., kidney, liver)

  • Establish optimal protocols before examining tissues with lower expression

  • Include appropriate negative controls

• Species considerations: Significant homology differences exist between human and other species' RIPK3. Commercial antibodies based on human sequences may not recognize RIPK3 in other species due to these differences .

How can I optimize RIPK3 antibody-based detection in virus-infected tissues?

Viral infection dramatically alters RIPK3 expression dynamics, requiring special consideration:

• Time-dependent expression: Research on nephropathogenic infectious bronchitis virus (NIBV) shows that RIPK3 upregulation in infected tissues follows a time-dependent pattern. Experimental design should include multiple time points post-infection .

• Multi-method approach: Combine complementary techniques for robust detection:

  • Western blot analysis for quantification

  • Real-time quantitative PCR for transcriptional changes

  • Immunofluorescence staining for localization changes

• Tissue-specific responses: NIBV infection significantly upregulated RIPK3 in trachea and kidney tissues, demonstrating tissue-specific responses to viral infection. Researchers should examine multiple relevant tissues rather than assuming uniform responses .

• Correlation with cell death markers: Co-stain for RIPK3 and cell death markers to establish functional relationships between RIPK3 upregulation and subsequent cellular outcomes .

How can RIPK3 mutants be utilized with antibodies to investigate kinase-dependent and independent functions?

RIPK3 mutants provide powerful tools for dissecting complex signaling mechanisms:

• Key kinase-dead mutants:

  • K51A: ATP-binding pocket mutation

  • D143N: Catalytic residue mutation (completely abolishes activity)

  • R142G: HRD motif mutation (substantially reduces activity)

• Experimental approaches:

  • In vitro kinase assays with purified recombinant RIPK3 kinase domains show D143N mutant entirely lacks detectable catalytic activity, while R142G has markedly reduced activity

  • Dimerization experiments using RIPK3 gyrase constructs containing these mutations demonstrate that kinase-inactive RIPK3 is unable to induce necroptosis

• Antibody applications with mutants:

  • Use total RIPK3 antibodies to confirm comparable expression of WT and mutant proteins

  • Apply phospho-specific antibodies to confirm loss of kinase activity

  • Investigate protein-protein interactions through co-immunoprecipitation

• Key finding: Research demonstrated that kinase-inactive or kinase-compromised RIPK3 gyrase is unable to induce necroptosis, even when expression levels are comparable to wild-type RIPK3 .

What role does RIPK3 play in viral infection and how can antibodies help elucidate these mechanisms?

RIPK3 has complex roles in viral infections that can be investigated using antibodies:

• Defense mechanisms:

  • RIPK3-dependent necroptosis can occur when caspases (and therefore apoptosis) are blocked by viral inhibitors

  • This cell death pathway contributes to the control of viral infections like Vaccinia virus

  • Pattern recognition receptors (PRRs) that detect viral PAMPs can initiate RIPK3 activation pathways

• Viral evasion strategies:

  • Some viruses produce inhibitors that target RIPK3

  • Murine Cytomegalovirus (MCMV) produces vIRA, which contains a RHIM domain that binds to RIPK3 and prevents necroptosis

  • MCMV with mutated RHIM domains (mutRHIM) cannot block RIPK3 activation, resulting in attenuation in wild-type mice but not in RIPK3-deficient mice

• Experimental approaches using antibodies:

  • Monitor RIPK3 expression levels during infection timecourse

  • Assess RIPK3 complex formation with other proteins (e.g., DAI, RIPK1)

  • Examine phosphorylation status as indicator of activation

• Case study: Research demonstrated that DAI (DNA-dependent activator of IRFs) contains RHIM domains and forms a complex with RIPK3 during MCMV infection, leading to necroptosis. This provides a mechanism for sensing viral DNA and triggering cell death .

How does RIPK3 deficiency impact immune responses and vaccination efficacy?

RIPK3 plays critical roles in immunity beyond its cell death functions:

• Vaccination responses:

  • Ripk3-/- mice show significantly reduced protection after vaccination with M2e (matrix protein 2 ectodomain) universal influenza A vaccine candidate

  • This occurs despite normal M2e-specific serum IgG levels, indicating the humoral immune response is not affected by RIPK3 deficiency

• Cellular immune responses:

  • Following influenza A virus challenge, M2e-vaccinated Ripk3-/- mice showed:

    • Decreased immune cell infiltrates in lungs

    • Increased accumulation of dead cells

    • Decreased presence of CD8+ T-cells

• Passive immunization efficacy:

  • While active vaccination protection was compromised in Ripk3-/- mice, passive transfer of anti-M2e monoclonal antibodies at higher doses completely protected Ripk3-/- mice against lethal influenza A virus infection

• Clinical implications:

  • Passive immunization strategies with monoclonal antibodies could be valuable for individuals with reduced vaccine efficacy

  • This might include patients treated with RIPK inhibitors for chronic inflammatory diseases

How can I address non-specific signals when using RIPK3 antibodies in immunofluorescence?

Non-specificity is a significant challenge with RIPK3 antibodies in immunofluorescence applications:

• Documented specificity issues:

  • Systematic evaluation of seven anti-human RIPK3 antibodies revealed several produced non-specific signals in immunofluorescence

  • Some antibodies showing specificity in Western blot may still yield non-specific signals in immunofluorescence (e.g., clone E1Z1D)

• Optimization strategies:

  • Titrate antibody concentration using positive and negative control samples

  • Modify fixation and permeabilization conditions (paraformaldehyde versus methanol fixation)

  • Test different blocking reagents to reduce background

  • Include genetic controls (RIPK3-deficient samples) whenever possible

• Alternative approaches:

  • Consider epitope-tagged RIPK3 constructs in transfectable systems

  • Use fluorescent protein-tagged RIPK3 for live-cell imaging

  • Apply proximity ligation assays for protein interaction studies

• Selection guidance: For critical immunofluorescence experiments, prioritize antibodies specifically validated for this application rather than relying solely on Western blot validation .

What approaches should I use for preparing and validating a RIPK3 polyclonal antibody?

For researchers developing custom RIPK3 antibodies, this systematic approach is recommended:

• Antigen design and expression:

  • Analyze target sequence homology across species

  • Design primers for amplifying target regions (e.g., 5′-CCGGAATTC GAAGTAGATATTTGGAGCAG-3′, 5′-CCCAAGCTT TGATGAGGTAAGGGATGT-3′)

  • Clone into an appropriate expression vector (e.g., pET-32a)

  • Express in bacterial system (BL21) with IPTG induction

• Protein purification:

  • For inclusion bodies, solubilize in 8M urea

  • Purify using Ni-NTA affinity column

  • Perform dialysis and renaturation

  • Verify fusion protein expression using anti-His-tag antibody

• Immunization and antibody production:

  • Immunize rabbits with purified protein

  • Collect serum after multiple immunizations

  • Determine antibody titer by ELISA (successful titers can reach 1:102,400)

• Validation:

  • Test specificity by Western blot against tissue panels

  • Compare with commercial antibodies

  • Validate in applications of interest (WB, IHC, IF)

• Key finding: Research demonstrated that successfully produced polyclonal antibodies could detect differential RIPK3 expression across tissues and changes in RIPK3 levels during viral infection .