RHBDD3 Antibody, HRP conjugated

What is RHBDD3 and why is it significant in immunological research?

RHBDD3 (Rhomboid Domain Containing 3) belongs to the rhomboid family of polytopic membrane serine proteases that play important roles in growth and development . Its significance in immunology stems from its function as a negative regulator of natural killer (NK) cell activation. Research using Rhbdd3-deficient mice has demonstrated that RHBDD3 is selectively upregulated in NK cells upon Toll-like receptor 3 (TLR3) stimulation and acts as a feedback inhibitor of TLR3-mediated NK cell activation . This regulatory role makes RHBDD3 an important target for studying immune regulation, particularly in contexts of inflammation and host defense against viral infections.

What are the primary research applications for RHBDD3 antibodies?

RHBDD3 antibodies are utilized in various research applications including:

• ELISA (Enzyme-Linked Immunosorbent Assay): Used for protein quantification and detection in solution

• Western Blot (WB): For protein detection and semi-quantitative analysis in tissue lysates and cell extracts

• Immunohistochemistry (IHC): For localization in tissue sections, including paraffin-embedded and frozen tissues

• Immunofluorescence (IF): For subcellular localization studies

These methodologies enable researchers to investigate RHBDD3 expression patterns, protein interactions, and functional roles in different experimental contexts.

What is the significance of HRP conjugation in RHBDD3 antibodies?

HRP (Horseradish Peroxidase) conjugation provides several methodological advantages:

• Direct detection: Eliminates the need for secondary antibodies in immunoassays

• Enhanced sensitivity: The enzymatic amplification provides stronger signal for low-abundance targets

• Reduced background: Fewer incubation steps decrease non-specific binding issues

• Versatility: Compatible with various substrates (TMB, DAB, etc.) for colorimetric or chemiluminescent detection

• Stability: HRP-conjugated antibodies typically maintain activity during long-term storage at -20°C

For RHBDD3 research, HRP conjugation is particularly valuable when studying its expression in tissues where levels may be variable or detecting its upregulation in NK cells following TLR3 stimulation .

How should I optimize ELISA protocols using HRP-conjugated RHBDD3 antibodies?

For optimal ELISA performance with HRP-conjugated RHBDD3 antibodies:

• Antibody titration: Determine optimal concentration through serial dilutions (typically starting at 1:100 to 1:2000)

• Blocking optimization: Test different blocking agents (BSA, casein, or commercial alternatives) to minimize background

• Sample preparation: Ensure consistent protein extraction methods across experimental groups

• Incubation conditions: Optimize temperature (4°C, room temperature) and duration (1-2 hours or overnight)

• Washing steps: Include 0.05% Tween-20 in wash buffer and perform at least 4-5 washes between steps

• Substrate selection: Choose appropriate HRP substrates based on sensitivity requirements

• Positive controls: Include recombinant RHBDD3 protein or samples from tissues known to express RHBDD3

The RHBDD3 antibody conjugated to HRP (like ABIN7140121) is specifically validated for ELISA applications , making it an excellent choice for quantitative analysis.

What are the recommended protocols for investigating RHBDD3's role in NK cell function?

Based on published research methodologies:

• NK cell isolation: Purify NK cells from spleen or liver using magnetic bead selection

• Stimulation conditions: Treat cells with poly(I:C) (10 μg/ml) to activate TLR3 signaling

• Time course analysis: Assess RHBDD3 expression at multiple timepoints (2h, 6h, 12h, 24h) following stimulation

• Protein interaction studies: Use co-immunoprecipitation to investigate RHBDD3 interaction with DAP12

• Functional assessment: Measure IFN-γ and granzyme B expression to correlate with RHBDD3 levels

• Cell-cell interaction analysis: Study NK cell interactions with accessory cells like dendritic cells and Kupffer cells

• Signaling pathway analysis: Assess MAPK activation in relation to RHBDD3 expression

Research has demonstrated that RHBDD3 increases quickly and significantly in NK cells after in vivo injection of poly(I:C) , providing a reliable experimental model.

What are the critical parameters for Western blot detection of RHBDD3?

For optimal Western blot detection of RHBDD3:

• Sample preparation: Use lysis buffers containing protease inhibitors to prevent degradation

• Protein loading: Load 20-50 μg of total protein per lane

• Gel percentage: Use 10-12% acrylamide gels for optimal resolution

• Transfer conditions: Transfer to PVDF membranes at 30V overnight at 4°C for best results

• Blocking: Block with 5% non-fat milk or BSA in TBST for 1-2 hours at room temperature

• Antibody dilution: Use RHBDD3 antibody at 1 μg/mL concentration (has been validated in rat lung tissue)

• Incubation: Incubate primary antibody overnight at 4°C with gentle rocking

• Detection system: Use appropriate HRP substrate with optimized exposure time

Western blot analysis should reveal a band at approximately 40 kDa, corresponding to the RHBDD3 protein .

How can I investigate the interaction between RHBDD3 and DAP12 in molecular detail?

To study the RHBDD3-DAP12 interaction:

• Co-immunoprecipitation: Immunoprecipitate with anti-DAP12 antibody followed by Western blot with HRP-conjugated RHBDD3 antibody

• Confocal microscopy: Visualize co-localization of RHBDD3 and DAP12 after poly(I:C) stimulation

• Proximity ligation assay: Detect protein-protein interactions in situ at single-molecule resolution

• Proteasome inhibition studies: Use MG132 to block protein degradation and assess DAP12 accumulation

• FRET analysis: Measure energy transfer between fluorescently labeled RHBDD3 and DAP12

• Domain mapping: Create deletion mutants to identify critical interaction domains

Research has shown that RHBDD3 interacts with DAP12 in poly(I:C)-activated NK cells and promotes its degradation, inhibiting MAPK activation in TLR3-triggered NK cells . This interaction requires cell-cell contact with accessory cells such as dendritic cells and Kupffer cells.

What methods can be employed to study RHBDD3's role in attenuating TLR3-triggered acute inflammation?

Based on published research approaches:

• In vivo models: Use Rhbdd3-deficient mice to study poly(I:C)-induced inflammation

• Liver inflammation assessment: Measure serum ALT and AST levels as indicators of liver damage

• Cytokine profiling: Quantify IFN-γ and IL-6 levels in serum and liver homogenates

• Cell population analysis: Assess NK cell accumulation in liver via flow cytometry

• Adoptive transfer experiments: Transfer Rhbdd3+/+ or Rhbdd3-/- NK cells into NK cell-depleted mice

• Cell depletion studies: Use clodronate liposomes to deplete Kupffer cells and assess impact on inflammation

• Histopathological analysis: Evaluate tissue damage and cellular infiltration in liver sections

Research has demonstrated that Rhbdd3 plays a critical role in attenuating TLR3-triggered acute inflammation by controlling NK cell activation and accumulation in liver and disrupting NK cell-Kupffer cell interaction .

How does RHBDD3 compare to other rhomboid family proteins in terms of structure and function?

Comparative analysis of RHBDD3 and other rhomboid proteins:

RHBDD3 was initially identified as a pituitary tumor apoptosis gene (PTAG), and its overexpression in AtT20 cells showed increased apoptotic activity and caspase activation in response to bromocriptine , distinguishing it from other family members.

What are common issues when using HRP-conjugated RHBDD3 antibodies and how can they be resolved?

Common problems and solutions:

• High background in immunoassays:

  • Increase blocking time or concentration

  • Optimize antibody dilution (typically 1:100-1:500)

  • Include additional washing steps with 0.1% Tween-20

  • Use fresh reagents and buffers

• Weak or no signal:

  • Verify sample preparation and protein integrity

  • Optimize antigen retrieval method for IHC/IF

  • Increase antibody concentration or incubation time

  • Use signal amplification systems (TSA, ABC method)

• Non-specific binding:

  • Validate antibody specificity with positive and negative controls

  • Pre-absorb antibody with tissue powder

  • Include peptide competition assays to confirm specificity

  • Use more stringent washing conditions

• Variable results between experiments:

  • Standardize tissue processing and storage conditions

  • Prepare fresh working solutions for each experiment

  • Include consistent positive control samples

How can I distinguish between specific and non-specific signals when detecting RHBDD3?

To distinguish specific from non-specific signals:

• Molecular weight verification: RHBDD3 should appear at approximately 40 kDa; bands at other weights may be non-specific

• Signal pattern analysis: Compare with known expression patterns across different tissues

• Control samples: Include RHBDD3-overexpressing and knockdown samples as positive and negative controls

• Peptide competition: Pre-incubation with the immunogenic peptide (aa 304-323 for some antibodies) should eliminate specific bands

• Cross-validation: Confirm findings with multiple antibodies targeting different RHBDD3 epitopes

• Species reactivity: Verify signal in appropriate species (human, mouse, rat) based on antibody specifications

• Cellular localization: Confirm expected subcellular distribution pattern

What considerations are important when interpreting RHBDD3 expression data in different experimental contexts?

Critical considerations for data interpretation:

• Cell type specificity: RHBDD3 is selectively up-regulated in NK cells upon TLR3 stimulation but not with IL-12/15 stimulation

• Temporal dynamics: Expression increases quickly after poly(I:C) stimulation, so timing of analysis is crucial

• Protein vs. mRNA levels: DAP12 protein levels are affected by RHBDD3 without changes in mRNA levels , highlighting the importance of protein-level analysis

• Cell-cell interactions: RHBDD3 function depends on cell-cell contact with accessory cells

• Pathological context: Expression is reduced in certain pituitary adenomas and colorectal tumors

• Functional correlation: Changes in RHBDD3 expression should correlate with NK cell activation markers (IFN-γ, granzyme B)

• Signaling pathway integration: Interpret in context of MAPK activation status

What are promising avenues for further investigation of RHBDD3 in immune regulation?

Emerging research directions:

• Single-cell analysis: Investigate RHBDD3 expression heterogeneity within NK cell populations

• Structural biology: Determine crystal structure of RHBDD3-DAP12 complex

• Therapeutic targeting: Develop small molecule modulators of RHBDD3 activity for inflammation control

• Cancer immunology: Explore the role of RHBDD3 in tumor-infiltrating NK cell function

• Viral immunity: Investigate RHBDD3's role in regulating NK cell responses to viral infections

• Systems biology: Map the complete RHBDD3 interactome in different immune cell types

• Translational research: Evaluate RHBDD3 as a biomarker for inflammatory conditions or cancer prognosis

How might RHBDD3 research contribute to understanding other immune regulatory pathways?

Broader implications for immunology:

• Negative feedback mechanisms: RHBDD3 exemplifies how NK cell activation is self-regulated through feedback inhibition

• Cell-cell communication: The dependence on accessory cells highlights the importance of cellular cross-talk in immune regulation

• Post-translational regulation: The promotion of DAP12 degradation by RHBDD3 demonstrates regulation beyond transcriptional control

• Signaling pathway integration: RHBDD3's effect on MAPK signaling connects it to broader cellular signaling networks

• Tissue-specific immune regulation: RHBDD3's role in liver inflammation points to tissue-specific regulatory mechanisms

• Therapeutic targets: Understanding RHBDD3 may inform development of new approaches to modulate inflammatory responses

• Evolutionary conservation: Studying rhomboid proteins across species may reveal fundamental immune regulatory principles