KLRC3 Antibody

What is KLRC3 and why is it important in immunological research?

KLRC3, also known as NKG2E, is a member of the killer cell lectin-like receptor subfamily that plays crucial roles in natural killer (NK) cell function and immune regulation. This receptor is significant in immunological research because it forms an intracytoplasmic complex with CD94 and DAP12, contributing to NK cell activation mechanisms . Understanding KLRC3 expression and function provides valuable insights into innate immunity, particularly NK cell-mediated responses against tumors and viral infections. Recent research has also linked KLRC3 expression to glycosylation-related gene expression in glioblastomas, suggesting broader implications in cancer biology . The study of KLRC3 through specific antibodies enables researchers to investigate these pathways in both healthy and pathological contexts.

How do I select the appropriate KLRC3 antibody for my research?

When selecting a KLRC3 antibody, consider four primary factors: target specificity, host species, clonality, and intended application. For specificity, determine whether you need an antibody targeting the N-terminal region (like ABIN2782166) or other specific amino acid sequences (such as AA 94-240 or AA 132-240) . Host species selection depends on your experimental design - rabbit polyclonal antibodies offer high sensitivity, while mouse monoclonal antibodies provide greater specificity and reproducibility . Regarding clonality, polyclonal antibodies recognize multiple epitopes and are beneficial for detecting low-abundance proteins, whereas monoclonal antibodies like clone 3D5 offer higher specificity for particular epitopes .

For application compatibility, review validation data carefully - some KLRC3 antibodies are specifically validated for Western blotting, while others are optimized for immunohistochemistry, immunofluorescence, or ELISA . Cross-check the antibody's reactivity with your experimental system (human, mouse, etc.) and examine available validation data showing performance in actual experimental conditions . For instance, ABIN7161649 has been validated for ELISA, IF, and IHC applications with human samples, while ABIN2782166 is validated for Western blotting with both human and mouse samples .

What are the alternative names for KLRC3 and why is this information important?

KLRC3 is known by several alternative names in scientific literature, including NK cell receptor E, NKG2-E type II integral membrane protein, NKG2-E-activating NK receptor, and NKG2E . Additional synonyms include KLRC2, NKG2-C, Nkg2e, and killer cell lectin like receptor C3 . This nomenclature diversity is critical information for researchers conducting comprehensive literature searches, as studies may reference the protein using different terminology. When searching databases like PubMed or designing experiments based on published protocols, awareness of these alternative names ensures you don't miss relevant research.

The use of alternative nomenclature also has practical implications when ordering antibodies or comparing results across studies. Some commercially available antibodies may be cataloged under different names, despite targeting the same protein . Understanding these naming conventions helps in correctly identifying relevant reagents and properly contextualizing your findings within the broader scientific literature, particularly when studying protein complexes where KLRC3/NKG2E interactions with partners like CD94 and DAP12 are reported .

What are the optimal protocols for using KLRC3 antibodies in immunohistochemistry?

For immunohistochemistry (IHC) applications with KLRC3 antibodies, optimal protocols involve careful tissue preparation, appropriate antigen retrieval, and specific dilution parameters. Begin with formalin-fixed, paraffin-embedded (FFPE) tissue sections cut to 4-6 μm thickness. After deparaffinization and rehydration, perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 15-20 minutes . This step is critical as inadequate antigen retrieval is a common cause of false negative results with KLRC3 detection.

For primary antibody incubation, dilution ratios vary by antibody source - for example, the KLRC3 antibody A46614 has demonstrated optimal results at a 1:20 dilution for human tissues like tonsil and cervical cancer samples . Incubate the primary antibody at 4°C overnight or for 1-2 hours at room temperature in a humidified chamber. Following washing with PBS-T (PBS containing 0.05% Tween-20), apply an appropriate secondary antibody system compatible with your detection method (HRP or AP-based) . For signal amplification in tissues with lower KLRC3 expression, consider employing biotin-streptavidin systems or tyramide signal amplification.

For negative controls, either omit the primary antibody or use a synthetic peptide competition assay, as demonstrated in the validation data for antibody A46614 . This control is particularly important for confirming signal specificity when examining tissues like tonsil or cervical cancer samples where KLRC3 expression patterns may vary. Counterstain with hematoxylin, dehydrate, and mount using a permanent mounting medium. Document magnification settings (typically 200x for initial assessment) when reporting results to facilitate comparison across studies .

How should I optimize Western blot conditions for KLRC3 detection?

Optimizing Western blot conditions for KLRC3 detection requires careful attention to sample preparation, electrophoresis parameters, and detection methods. Begin by extracting proteins using a lysis buffer containing protease inhibitors to prevent KLRC3 degradation. For membrane-associated proteins like KLRC3, include detergents such as 1% NP-40 or 0.5% Triton X-100 in your lysis buffer to ensure effective solubilization . Quantify protein concentration using BCA or Bradford assays to ensure equal loading across samples.

When preparing samples, use reducing conditions with DTT or β-mercaptoethanol, as KLRC3 detection may be influenced by proper denaturation of its tertiary structure. Load 20-50 μg of total protein per lane on a 10-12% SDS-PAGE gel, which provides optimal resolution for KLRC3 (molecular weight range). After electrophoresis, transfer proteins to PVDF membranes (rather than nitrocellulose) for enhanced protein binding and signal-to-noise ratio .

For primary antibody incubation, KLRC3 antibodies targeting the N-terminal region (like ABIN2782166) typically perform well at 1:500 to 1:1000 dilutions in 5% BSA or non-fat milk blocking solution . Incubate overnight at 4°C with gentle rocking. After thorough washing with TBS-T (at least 3 x 10 minutes), apply HRP-conjugated secondary antibodies at 1:5000 dilution for 1 hour at room temperature. Use enhanced chemiluminescence (ECL) detection with exposure times optimized based on expression levels - typically starting with 30-second exposures and adjusting as needed. Include positive control lysates during optimization, as these have been used to validate antibodies like ABIN2782166 . For multiplex detection, consider using fluorescent secondary antibodies and specialized imaging systems to simultaneously detect KLRC3 and interaction partners like CD94.

What controls should be incorporated when using KLRC3 antibodies in research applications?

Incorporating appropriate controls when using KLRC3 antibodies is essential for result validation and experimental rigor. For positive controls, use tissues or cell lines with confirmed KLRC3 expression - human tonsil tissue serves as an excellent positive control for immunohistochemistry applications as demonstrated in validation studies . NK cell lines such as NK-92 or primary NK cells are also suitable positive controls for Western blot and flow cytometry applications. These positive controls confirm that absence of signal results from lack of target protein rather than technical issues.

Negative controls should include samples known to lack KLRC3 expression or experimental conditions that specifically block antibody binding. For instance, peptide competition assays, where the antibody is pre-incubated with the immunizing peptide (as shown in the validation data for antibody A46614), effectively demonstrate specificity by blocking binding to the target epitope . For polyclonal antibodies, using pre-immune serum from the same animal can help distinguish between specific and non-specific binding.

For advanced experiments, include isotype controls matching the primary antibody's host species and immunoglobulin class but lacking specificity for the target. This controls for potential Fc receptor binding or other non-specific interactions. When performing knockdown or knockout studies, include KLRC3-depleted samples as gold-standard negative controls. For antibodies with cross-reactivity between human and mouse KLRC3 (like ABIN2782166 with 100% human and 79% mouse predicted reactivity), use species-specific positive controls to confirm the cross-reactivity in your experimental system . Additionally, when studying complex formation between KLRC3/NKG2E and partners like CD94/DAP12, include controls for co-immunoprecipitation experiments to verify the specificity of detected interactions .

How can I address non-specific binding issues when using KLRC3 antibodies?

Non-specific binding is a common challenge when using KLRC3 antibodies that can be addressed through several methodological refinements. First, optimize your blocking protocol by testing different blocking agents - 5% BSA often provides superior results compared to non-fat milk for membrane proteins like KLRC3. Extend blocking time to 2 hours at room temperature or overnight at 4°C to ensure complete blocking of non-specific binding sites . For Western blots, increase the concentration of Tween-20 in washing buffers from 0.05% to 0.1% and perform more extensive washing steps (5 x 5 minutes instead of 3 x 5 minutes).

If background persists, dilute the primary antibody further than recommended - for instance, if using KLRC3 antibody A46614 with a standard dilution of 1:20 for IHC, try 1:30 or 1:40 . Additionally, reduce incubation time or temperature for the primary antibody. For immunofluorescence applications, include an autofluorescence quenching step using sodium borohydride or commercial quenching reagents before the blocking step.

For polyclonal antibodies that show cross-reactivity, consider pre-adsorption against tissues or cell lysates from species with high sequence homology but lacking the specific epitope. This is particularly useful when using antibodies like ABIN2782166 that have predicted reactivity to both human (100%) and mouse (79%) KLRC3 . In extreme cases where non-specific binding persists despite these measures, consider antibody purification techniques such as affinity purification against the immunogen peptide, similar to the purification method used for ABIN2782166 . Document all optimization steps in your experimental methods to provide a clear pathway for reproducibility.

What are the key validation criteria for ensuring KLRC3 antibody specificity in my experiments?

Validating KLRC3 antibody specificity requires a multi-faceted approach employing several complementary techniques. Begin with peptide competition assays, where pre-incubation of the antibody with its specific immunogenic peptide should substantially reduce or eliminate signal in your application, as demonstrated in the validation data for antibody A46614 in IHC applications . This confirms that the antibody is binding to its intended target epitope rather than producing non-specific signals.

Western blot validation should demonstrate a single predominant band at the expected molecular weight for KLRC3, with minimal or no additional bands. Compare the observed molecular weight with theoretical predictions, accounting for potential post-translational modifications like glycosylation that might affect migration patterns. For antibodies targeting specific regions, such as ABIN2782166 (N-terminal) or antibodies targeting amino acids 94-240 or 132-240, validation should include testing against recombinant proteins containing these specific domains .

For greater confidence in specificity, implement genetic approaches: test the antibody in KLRC3 knockout or knockdown systems where signal should be significantly reduced or absent. This gold-standard approach confirms that the observed signal is truly from KLRC3 rather than cross-reactive proteins. Additionally, validate across multiple applications (WB, IHC, IF, ELISA) when possible, as consistent results across techniques strongly support specificity .

Cross-validate with multiple antibodies targeting different epitopes of KLRC3 - concordant results from different antibodies (like comparing results from ABIN561610 targeting AA 132-240 and ABIN2782166 targeting the N-terminus) provide strong evidence for specific detection . Finally, confirm that the antibody can detect known biological relationships, such as the reported intracytoplasmic complex formation between KLRC3/NKG2E, CD94, and DAP12 . Document all validation results thoroughly, including both positive findings and any limitations observed during the validation process.

How do I interpret contradictory results from different KLRC3 antibodies?

Interpreting contradictory results from different KLRC3 antibodies requires systematic analysis of several factors that might influence antibody performance. First, examine epitope differences - antibodies targeting different regions of KLRC3, such as N-terminal regions (ABIN2782166) versus other domains (AA 94-240 or AA 132-240), may produce different results if epitope accessibility varies across experimental conditions or if post-translational modifications mask certain epitopes . This is particularly relevant when comparing results between native and denatured conditions, as conformational epitopes may be lost in denaturing applications.

Consider clonality differences - monoclonal antibodies like clone 3D5 recognize single epitopes with high specificity but may fail if that specific epitope is unavailable, while polyclonal antibodies recognize multiple epitopes, offering greater detection probability but potentially higher background . Application-specific optimization is also critical - an antibody validated extensively for IHC (like A46614) may not perform optimally in Western blotting without specific protocol adjustments .

When faced with contradictory results, implement a hierarchical validation approach: first, repeat experiments under identical conditions to confirm reproducibility of the contradictory findings. Then, test both antibodies in a system with controlled KLRC3 expression (overexpression or knockdown) to determine which antibody more accurately reflects actual protein levels. Cross-validate with non-antibody methods such as mRNA expression analysis or mass spectrometry to provide independent confirmation of KLRC3 presence and abundance.

Create a comparison table documenting key parameters:

This structured approach helps identify whether contradictions arise from technical factors (application-specific performance), biological factors (epitope availability in different contexts), or reagent-specific limitations that need to be acknowledged in result interpretation and reporting .

How can KLRC3 antibodies be optimized for studying protein-protein interactions in NK cell signaling complexes?

Optimizing KLRC3 antibodies for protein-protein interaction studies requires techniques that preserve native protein complexes while maintaining detection sensitivity. For co-immunoprecipitation (Co-IP) experiments investigating the reported KLRC3/NKG2E complex formation with CD94 and DAP12, use lysis buffers with mild detergents (0.5% NP-40 or 0.1% digitonin) to preserve membrane protein associations . Pre-clear lysates with appropriate control IgG conjugated to the same beads used for immunoprecipitation to reduce non-specific binding. Select antibodies with proven specificity for native conformations - for instance, rabbit polyclonal antibodies may perform better than mouse monoclonal antibodies in Co-IP applications due to recognition of multiple epitopes.

For proximity ligation assays (PLA) to visualize KLRC3 interactions in situ, use antibody pairs raised in different host species (e.g., rabbit anti-KLRC3 and mouse anti-CD94) to enable species-specific secondary antibody recognition. Optimize fixation conditions to preserve both membrane architecture and epitope accessibility - typically, 2-4% paraformaldehyde for 10-15 minutes maintains this balance for membrane proteins like KLRC3 . Consider using membrane-permeabilizing agents like 0.1% saponin rather than stronger detergents like Triton X-100 that might disrupt membrane protein complexes.

For bimolecular fluorescence complementation (BiFC) or FRET-based approaches, conjugate KLRC3 antibodies directly with appropriate fluorophores using commercial conjugation kits, maintaining a dye-to-antibody ratio of 2-4 for optimal signal-to-noise ratios. When designing multiplexed detection systems, select fluorophores with minimal spectral overlap and use appropriate compensation controls. Advanced super-resolution microscopy techniques (STORM, PALM) can further enhance visualization of KLRC3-containing complexes at the plasma membrane, requiring specialized fluorophore selection and sample preparation protocols that preserve nanoscale protein distributions while maintaining antibody accessibility to targeted epitopes .

What strategies can be employed for multiplex detection of KLRC3 alongside other NK cell receptors?

Multiplex detection of KLRC3 alongside other NK cell receptors requires careful antibody selection and optimization of detection systems to minimize cross-reactivity while maximizing signal specificity. For immunofluorescence and flow cytometry applications, select primary antibodies raised in different host species (e.g., rabbit anti-KLRC3 and mouse anti-CD94) to enable use of species-specific secondary antibodies with distinct fluorophores . When limited by available host species, use directly conjugated primary antibodies with minimal spectral overlap. For example, combine Alexa Fluor 488-conjugated anti-KLRC3 with Alexa Fluor 647-conjugated anti-CD94 and PE-conjugated anti-DAP12 to simultaneously visualize all three components of the reported intracytoplasmic complex .

For multiplexed immunohistochemistry, implement tyramide signal amplification (TSA) protocols that allow sequential detection of multiple targets on the same tissue section. This approach involves applying the first primary antibody, detecting with HRP-conjugated secondary antibody and TSA, followed by heat-mediated antibody stripping before applying the next primary antibody. This method can be particularly valuable when examining KLRC3 expression in complex tissues like tonsil or cervical cancer samples, where multiple cell types express various NK receptors in close proximity .

For quantitative proteomics approaches, implement sequential immunoprecipitation strategies where one receptor (e.g., KLRC3) is immunoprecipitated first, followed by elution under mild conditions and subsequent immunoprecipitation of interaction partners. Alternatively, employ mass cytometry (CyTOF) using metal-tagged antibodies against KLRC3 and other NK receptors, which eliminates issues of spectral overlap encountered in conventional flow cytometry. This approach is particularly valuable for comprehensive phenotyping of NK cell receptor expression patterns in complex samples like tumor-infiltrating lymphocytes, where simultaneous detection of 30+ markers can reveal novel receptor co-expression patterns .

How can KLRC3 antibodies be applied in studying the role of this receptor in cancer immunosurveillance?

KLRC3 antibodies offer valuable tools for investigating this receptor's role in cancer immunosurveillance through several advanced applications. For tumor tissue analysis, implement multiplex immunohistochemistry using antibodies like A46614 (which has been validated on cervical cancer tissue) to simultaneously visualize KLRC3-expressing NK cells, tumor cells, and other immune populations within the tumor microenvironment . Optimize staining protocols to preserve tissue architecture while enabling quantitative assessment of NK cell infiltration patterns and their correlation with KLRC3 expression levels. This approach provides spatial context for understanding NK cell-tumor cell interactions mediated by KLRC3/NKG2E.

For functional studies, combine KLRC3 antibodies with cytotoxicity assays to determine how receptor blockade or activation affects NK cell-mediated killing of tumor cells. Use Fab fragments or non-activating KLRC3 antibodies to block receptor function without inducing antibody-dependent cellular cytotoxicity (ADCC), providing clean experimental systems for studying receptor-specific effects. Complement these approaches with flow cytometry-based degranulation assays (CD107a exposure) and cytokine production measurements to comprehensively assess how KLRC3 modulates NK cell functional responses to various tumor types.

To investigate potential correlations between KLRC3 expression and glycosylation patterns in cancer cells (building on reported relationships in glioblastoma research), implement dual staining approaches with KLRC3 antibodies and glycan-binding lectins or glycosylation-specific antibodies . This approach can reveal how tumor-specific glycosylation modifications might influence KLRC3-mediated recognition. For translational applications, develop immunomonitoring protocols using KLRC3 antibodies to assess NK cell receptor expression in patient samples before and after immunotherapy, potentially identifying KLRC3 expression patterns that correlate with treatment response. These applications collectively enable systematic investigation of how KLRC3/NKG2E contributes to cancer immunosurveillance mechanisms and how this knowledge might be leveraged for immunotherapeutic strategies .