Klra3 Antibody

What is Klra3 and what is its biological significance?

Klra3 (also known as Ly49C) is a member of the LY49 family of receptors expressed on Natural Killer (NK) cells that bind to major histocompatibility complex (MHC) class 1. Klra3 functions as an inhibitory receptor, containing immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic region. This receptor recognizes peptide-receptive H-2Kb molecules and plays a crucial role in regulating NK cell activity through inhibitory signaling pathways. Understanding Klra3 function is essential for research into immune surveillance, self-tolerance, and NK cell-mediated cytotoxicity mechanisms .

What types of Klra3 antibodies are available for research applications?

Several types of Klra3 antibodies are available for research purposes, varying in host species, clonality, and conjugation:

The choice between these antibodies depends on the specific experimental requirements, including detection method, target species, and the biological question being addressed .

What are the common reactivity profiles of Klra3 antibodies?

Most commercially available Klra3 antibodies demonstrate reactivity with mouse samples, while some also cross-react with rat tissues. The monoclonal antibody 14B11 has a defined reactivity profile, recognizing multiple members of the Ly-49 family including Ly-49C (Klra3), Ly-49F, Ly-49I, and Ly-49H, but notably does not react with Ly-49A, B, D, or G. This broader reactivity profile can be advantageous for certain applications but requires careful interpretation when specific detection of Klra3 alone is desired. Each antibody should be evaluated based on the specific experimental requirements and validated in the researcher's model system .

What are the optimal protocols for Western blot detection of Klra3?

For optimal Western blot detection of Klra3:

• Sample preparation: Prepare cell or tissue lysates in RIPA buffer supplemented with protease inhibitors. NK cell-enriched samples from spleen or lymph nodes generally provide sufficient Klra3 expression.

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

• Separation conditions: Use 10-12% SDS-PAGE gels for optimal resolution.

• Transfer parameters: Transfer to PVDF membranes at 100V for 1 hour or 30V overnight.

• Blocking: Block with 5% non-fat dry milk in TBST for 1 hour at room temperature.

• Primary antibody: Dilute polyclonal Klra3 antibody to 2 μg/mL in blocking buffer and incubate overnight at 4°C. This concentration has been validated for mouse samples in Western blot applications.

• Detection: Use appropriate HRP-conjugated secondary antibodies and enhanced chemiluminescence detection.

The expected molecular weight for Klra3 is approximately 45-50 kDa, though glycosylation can result in higher apparent molecular weights. Non-reducing conditions may better preserve conformational epitopes for some antibodies .

How should researchers optimize flow cytometry protocols with Klra3 antibodies?

For flow cytometry applications using Klra3 antibodies:

• Sample preparation: Prepare single-cell suspensions from spleen, lymph nodes, or other tissues containing NK cells. Use RBC lysis buffer if necessary.

• Cell concentration: Adjust to 1-5 × 10^6 cells per 100 μL for staining.

• Fc receptor blocking: Pre-incubate cells with anti-CD16/CD32 to prevent non-specific binding.

• Antibody titration: Titrate the 14B11 monoclonal antibody, with recommended concentration of ≤0.125 μg per test (where a test is defined as the amount of antibody that will stain a cell sample in a final volume of 100 μL).

• Multi-parameter considerations: When designing panels, consider that PE-conjugated antibodies work well with the 14B11 clone, with excitation at 488-561 nm and emission at 578 nm.

• Gating strategy: Use NK cell markers (NK1.1, CD3-) in combination with Klra3 for proper identification of Klra3+ NK cells.

• Controls: Include FMO (fluorescence minus one) controls and isotype controls appropriate to the host species and antibody class.

For enhanced results, researchers should consider that the 14B11 antibody reportedly enhances the redirected lysis of FcR+ target cells by IL-2-activated NK cells, which may be relevant for functional experiments .

How can cross-reactivity with other LY49 receptors be addressed in experimental design?

Cross-reactivity with other LY49 family members is a significant consideration when working with Klra3 antibodies. Several approaches can minimize or account for this issue:

• Antibody selection: Choose antibodies validated for specificity whenever possible. The 14B11 clone has known cross-reactivity with Ly-49F, I, and H, but not with Ly-49A, B, D, or G.

• Knockout controls: When available, use Klra3-knockout tissues/cells as negative controls.

• Competitive blocking: Pre-incubate with recombinant Klra3 protein to demonstrate specificity.

• Multiple antibody approach: Use multiple antibodies targeting different epitopes of Klra3 to confirm results.

• Complementary techniques: Combine antibody-based detection with other methods like qPCR for Klra3 mRNA.

• Multi-parameter flow cytometry: Use additional NK receptor markers to distinguish between Klra3 and other Ly49 family members.

• Western blot verification: Confirm specificity by molecular weight in Western blot applications.

These strategies, used in combination, can significantly improve confidence in the specificity of Klra3 detection despite the challenge of cross-reactivity within the Ly49 family .

What are the considerations for storage and handling of Klra3 antibodies to maintain optimal activity?

Proper storage and handling are crucial for maintaining antibody performance:

• Storage temperature: Store Klra3 antibodies at 4°C for short-term (up to three months) or at -20°C for long-term storage (up to one year).

• Aliquoting: Upon receipt, prepare small working aliquots to avoid repeated freeze-thaw cycles, which can degrade antibody quality.

• Buffer conditions: Klra3 antibodies are typically supplied in PBS containing 0.02% sodium azide as a preservative. Some formulations may contain 50mM Sodium Borate or other stabilizers.

• Conjugated antibodies: Store fluorophore-conjugated antibodies (such as PE, FITC, or Janelia Fluor 525) in the dark to prevent photobleaching.

• Temperature control: When working with the antibody, maintain cold chain by keeping on ice during experimental procedures.

• Centrifugation: Briefly centrifuge antibody vials before opening to collect liquid at the bottom.

• Contamination prevention: Use sterile technique when handling antibodies to prevent microbial contamination.

Following these guidelines will help ensure consistent performance and extend the useful life of Klra3 antibodies in laboratory settings .

What controls should be included in experiments using Klra3 antibodies?

A comprehensive set of controls is essential for reliable interpretation of results when using Klra3 antibodies:

Properly implemented controls increase confidence in results and facilitate troubleshooting when unexpected outcomes occur .

How can researchers troubleshoot weak or non-specific signals with Klra3 antibodies?

When encountering issues with Klra3 antibody performance, consider the following troubleshooting approaches:

For weak signals:

• Antibody concentration: Increase antibody concentration incrementally (begin with 2 μg/mL for Western blot and titrate upward if necessary).

• Incubation conditions: Extend primary antibody incubation time or use higher temperature.

• Enhanced detection: Use more sensitive detection systems (e.g., enhanced chemiluminescence for WB).

• Sample enrichment: Enrich for NK cells before analysis to increase target concentration.

• Reduced washing: Decrease wash stringency to preserve weak signals.

For non-specific signals:

• Blocking optimization: Increase blocking agent concentration or try alternative blocking agents.

• Antibody dilution: Use more dilute antibody solutions to reduce non-specific binding.

• Wash stringency: Increase number and duration of washes.

• Cross-adsorption: Pre-adsorb antibody with tissues/cells lacking Klra3 but expressing other Ly49 family members.

• Buffer optimization: Adjust salt concentration or add detergents to reduce non-specific interactions.

• Alternative antibody: Try another Klra3 antibody targeting a different epitope.

Remember that Klra3 antibodies may cross-react with other LY49 receptors, so apparent non-specific signals may actually represent detection of related proteins .

What factors influence Klra3 expression and detection in different experimental systems?

Several factors can impact Klra3 expression and detection:

• Mouse strain variation: Klra3 expression differs significantly between mouse strains, with C57BL/6 mice expressing higher levels than some other strains.

• Activation state: NK cell activation can alter Klra3 expression levels and surface localization.

• Tissue source: Expression levels vary between different tissues and lymphoid organs.

• Age and development: NK cell receptor expression changes during development and aging.

• Cytokine environment: Exposure to cytokines like IL-2, IL-15, and IL-12 can modulate Klra3 expression.

• Viral infection: Certain viral infections can downregulate or alter Klra3 expression.

• Sample preparation: Enzymatic tissue dissociation methods may cleave surface receptors, affecting detection.

• Fixation effects: Some fixation methods can mask epitopes recognized by certain antibodies.

When designing experiments, researchers should consider these factors and standardize conditions across experimental groups. For comparing results between different studies, careful attention to these variables is essential .

How can Klra3 antibodies be used to study NK cell inhibitory function?

Klra3 antibodies are valuable tools for investigating NK cell inhibitory functions through several methodologies:

• Receptor blocking studies: The 14B11 monoclonal antibody can be used to block Klra3 interaction with MHC class I molecules, allowing researchers to observe the effects of removing this inhibitory signal on NK cell function. This approach reveals the contribution of Klra3 to NK cell self-tolerance and activation thresholds.

• Redirected lysis assays: The 14B11 antibody has been reported to enhance the redirected lysis of FcR+ target cells by IL-2-activated NK cells, providing a functional readout of receptor engagement.

• Phosphorylation analysis: Following Klra3 engagement, researchers can use phospho-specific antibodies to detect activation of inhibitory signaling pathways, particularly those involving SHP-1 and SHP-2 phosphatases that are recruited to the ITIM motifs.

• Co-immunoprecipitation: Using Klra3 antibodies for immunoprecipitation allows for the identification of protein interaction partners involved in inhibitory signaling cascades.

• FACS-based cytotoxicity assays: By labeling NK cells with Klra3 antibodies, researchers can correlate receptor expression with cytotoxic function at the single-cell level.

These applications provide insights into how Klra3 contributes to the complex regulation of NK cell responses in health and disease .

What approaches can be used to quantify Klra3 expression levels in research samples?

Quantitative analysis of Klra3 expression can be performed using several complementary approaches:

For the most robust quantification, researchers should use flow cytometry with carefully titrated antibodies and appropriate controls. When using the 14B11 clone, a concentration of ≤0.125 μg per test is recommended for optimal signal-to-noise ratio. Standardized beads can be used to convert arbitrary fluorescence units to absolute numbers of receptors per cell .

How can Klra3 antibodies be integrated into multi-parameter flow cytometry panels?

Designing effective multi-parameter panels incorporating Klra3 antibodies requires careful consideration of several factors:

• Fluorophore selection: The PE-conjugated 14B11 antibody (excitation: 488-561 nm; emission: 578 nm) works well with blue, green, or yellow-green lasers. Consider brightness hierarchy when designing panels, with PE being a relatively bright fluorophore suitable for targets with moderate expression levels.

• Panel design strategy:

  • Include CD3 (negative) and NK1.1 or NKp46 (positive) to identify NK cells

  • Add CD11b and CD27 to distinguish NK developmental subsets

  • Include additional activating (NKG2D, NKp46) and inhibitory receptors (NKG2A, other Ly49 members) for comprehensive phenotyping

  • Consider functional markers (IFN-γ, perforin, granzymes) for functional correlation

• Spectral overlap considerations: When using PE-conjugated Klra3 antibodies, avoid or compensate for fluorophores with significant spectral overlap, such as PE-Cy5 or PE-Cy7 on critical markers.

• Sequential gating strategy:

  • First gate on viable cells (using viability dye)

  • Gate on lymphocytes based on FSC/SSC

  • Identify NK cells as CD3-/NK1.1+ or CD3-/NKp46+

  • Analyze Klra3 expression on NK cell subsets

• Controls:

  • FMO control for Klra3 to set positive gates

  • Compensation controls for all fluorophores

  • Isotype control matched to host species and antibody class

This approach allows for comprehensive characterization of Klra3 expression in the context of NK cell heterogeneity and function .

What are the emerging research applications for Klra3 antibodies in cancer immunotherapy studies?

Klra3 antibodies are increasingly valuable in cancer immunotherapy research, with several emerging applications:

• NK cell exhaustion phenotyping: Characterizing changes in Klra3 expression on tumor-infiltrating and peripheral NK cells to understand NK cell dysfunction in cancer.

• Checkpoint inhibition strategies: Exploring Klra3 blockade as a potential checkpoint inhibition approach, similar to PD-1/PD-L1 blockade in T cells.

• CAR-NK engineering: Using Klra3 expression data to select optimal NK cell subsets for chimeric antigen receptor (CAR) engineering.

• MHC-I downregulation studies: Investigating how cancer-associated MHC-I downregulation affects Klra3-mediated inhibitory signaling in the tumor microenvironment.

• Combination therapy biomarkers: Evaluating Klra3 expression as a potential biomarker for response to immunotherapies, including those targeting other immune checkpoints.

• NK cell education: Studying how Klra3 interactions with MHC-I molecules during NK cell development (education) impact anti-tumor responses.

• Ex vivo expansion protocols: Optimizing NK cell expansion protocols by monitoring Klra3 expression to maintain functional NK cells for adoptive transfer.

These applications highlight the importance of high-quality Klra3 antibodies in advancing our understanding of NK cell biology in cancer and developing novel immunotherapeutic approaches .

What are the limitations of current Klra3 antibodies and how might they be addressed in future research?

Current Klra3 antibodies face several limitations that future research could address:

• Cross-reactivity: Many available antibodies cross-react with other Ly49 family members. Development of more specific monoclonal antibodies or recombinant antibody fragments with enhanced specificity would improve research accuracy.

• Limited species reactivity: Most antibodies recognize mouse Klra3, with limited options for other species. Expanding the range of species-specific antibodies would facilitate comparative studies.

• Application restrictions: Many antibodies are validated only for specific applications (e.g., Western blot, flow cytometry). More comprehensive validation across multiple applications would increase versatility.

• Epitope mapping: Limited information on exact epitope binding regions restricts understanding of potential functional interference. Detailed epitope mapping would enhance interpretation of blocking studies.

• Functional grade limitations: Few antibodies are specifically validated for functional studies like receptor blocking. Development of antibodies optimized for functional applications would expand research capabilities.

Future directions may include development of recombinant antibody technologies, single-domain antibodies (nanobodies), and expanded validation across applications and species to address these limitations .

How do researchers integrate Klra3 antibody data with other NK cell receptor analyses for comprehensive understanding?

Comprehensive NK cell research requires integration of Klra3 data with broader receptor analyses:

• Multiparameter analysis strategies: Modern flow cytometry and mass cytometry (CyTOF) allow simultaneous assessment of Klra3 alongside multiple activating and inhibitory receptors, providing insights into receptor coexpression patterns and functional correlations.

• Systems biology approaches: Computational modeling of receptor interactions and signaling networks helps predict how Klra3 functions within the broader context of NK cell regulation.

• Single-cell technologies: Combining Klra3 protein detection with single-cell RNA-seq or CITE-seq allows correlation of receptor expression with transcriptional profiles at unprecedented resolution.

• Functional correlation: Relating Klra3 expression to cytotoxicity, cytokine production, and proliferation provides context for interpreting receptor expression data.

• Receptor pairing analysis: Understanding how Klra3 expression correlates with other inhibitory and activating receptors on the same cell provides insights into the "rheostat" model of NK cell activation.

• Spatial analysis: Techniques like imaging mass cytometry and multiplexed immunofluorescence preserve spatial information about Klra3 expression in tissue contexts.