EFHD2 Antibody, HRP conjugated

What is EFHD2 and why is it important in research?

EFHD2 (also known as Swiprosin-1) is a calcium-binding adaptor protein of the EF hand family that plays important roles in neuronal and immune cell Ca²⁺ signaling. This highly conserved protein has been proposed to be involved in B cell signaling pathways and various neuropathological disorders, making it a subject of interest across multiple research disciplines . EFHD2 contains a distinctive N-terminal low complexity region that differentiates it from its homologous protein EFHD1 (Swiprosin-2), with which it shares approximately 69.7% sequence identity and similar predicted protein structure . In B cells, EFHD2 functions as a negative regulator of germinal center-dependent humoral immunity, with particular implications for IgE generation in type 2 immune responses . Additionally, recent research has demonstrated that EFHD2 can co-aggregate with both monomeric and filamentous tau proteins, suggesting potential involvement in neurodegenerative pathways .

How can EFHD2 antibodies distinguish between EFHD1 and EFHD2 proteins?

EFHD2-specific monoclonal antibodies recognize epitopes within the N-terminal low complexity region of EFHD2, which contains immunodominant sequences that differ significantly from EFHD1 . According to experimental validation studies, anti-EFHD2 monoclonal antibodies (such as A4.15.28, A4.15.48, A4.18.18, and E7.20.23) can successfully recognize both murine and human EFHD2 but demonstrate no cross-reactivity with murine EFHD1, even in overexpression systems . The specificity of these antibodies has been rigorously confirmed through multiple methodologies including Western blotting, immunoprecipitation, and flow cytometry analyses using cells lacking EFHD2 expression via shRNA silencing, cells with reconstituted EFHD2 expression, and cells expressing EFHD2 deletion mutants . Differential expression patterns further support this distinction - for example, EFhd1 protein expression has been detected exclusively in pro-B cells (38B9) among various transformed B cell lines, whereas EFHD2 is expressed across multiple B cell developmental stages including pro-B, pre-B, activated immature and mature B cells, and plasma cells .

What are the optimal sample preparation methods for EFHD2 detection using HRP-conjugated antibodies?

For optimal EFHD2 detection using HRP-conjugated antibodies, sample preparation methods should be tailored to the specific application while preserving the protein's native structure and epitope accessibility. When preparing cell lysates for Western blot analysis, researchers should lyse cells using non-denaturing conditions with buffers containing 1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl (pH 7.4), and protease inhibitor cocktails to maintain protein integrity . For immunohistochemistry applications, tissues should be fixed with 4% paraformaldehyde followed by paraffin embedding or frozen sectioning, with subsequent permeabilization using 0.1-0.5% Triton X-100 . When preparing samples for flow cytometry, cells should be fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin in PBS/1% BSA, and blocked with 2% FBS prior to antibody staining . It is crucial to include appropriate controls in all experiments, such as EFHD2-deficient cells (e.g., WEHI231.shEFhd2 cells) or tissues from EFHD2 knockout mice, to validate staining specificity and eliminate false-positive signals that might arise from non-specific binding .

How can researchers quantify EFHD2 expression levels in different cell populations?

Flow cytometry with anti-EFHD2 antibodies provides a robust method for quantifying EFHD2 expression levels across different cell populations with high sensitivity and cellular resolution. Researchers have successfully established flow cytometric protocols that can quantify EFHD2 expression in a linear manner over two log scales, enabling precise comparative analyses between different cell types or experimental conditions . When implementing this approach, cells should be fixed, permeabilized, and stained with titrated concentrations of anti-EFHD2 antibodies (such as A4.18.18), with careful optimization of antibody concentration to ensure detection within the linear range . To validate specificity, researchers should include controls such as isotype-matched antibodies and competitive blocking experiments using recombinant EFHD2 protein . This methodology has revealed significant biological insights, such as the approximately five-fold higher expression of EFHD2 in human monocytes compared to B cells in peripheral blood mononuclear cells (PBMCs) . For multi-parameter analysis, researchers can combine EFHD2 staining with additional markers to simultaneously interrogate EFHD2 expression across different immune cell subsets, enabling complex phenotypic characterization in both healthy and disease states.

What strategies improve Western blot detection of EFHD2 using HRP-conjugated antibodies?

To improve Western blot detection of EFHD2 using HRP-conjugated antibodies, researchers should implement several optimization strategies addressing sample preparation, transfer efficiency, and signal enhancement. Since EFHD2 migrates at approximately 26-30 kDa, researchers should optimize gel percentage (12-15% polyacrylamide) to achieve optimal protein separation in this molecular weight range . When preparing samples, complete denaturation can be achieved using SDS-PAGE loading buffer containing 5% β-mercaptoethanol with heating at 95°C for 5 minutes, although in some cases, mild denaturation (heating at 65°C) may better preserve certain epitopes . After electrophoresis, efficient protein transfer should be performed using PVDF membranes (rather than nitrocellulose) with 20% methanol-containing transfer buffer at 100V for 1 hour or 30V overnight at 4°C . Blocking should be performed with 5% non-fat dry milk in TBST (TBS with 0.05% Tween-20) for 1 hour at room temperature, followed by overnight incubation with HRP-conjugated anti-EFHD2 antibody at 4°C . For enhanced sensitivity with minimal background, researchers should optimize antibody dilution (typically 1:1000 to 1:5000), incorporate additional washing steps (at least 3×10 minutes with TBST), and utilize enhanced chemiluminescence (ECL) detection systems with extended exposure times if necessary.

How can EFHD2 antibodies be used to investigate subcellular localization patterns?

EFHD2 antibodies can effectively reveal the protein's distinct subcellular distribution patterns through immunofluorescence microscopy, subcellular fractionation, and co-localization studies with organelle markers. Immunofluorescence studies using anti-EFHD2 monoclonal antibodies have identified two major EFHD2 pools in B cells: one at the plasma membrane and another in intracellular vesicular structures . To optimize subcellular visualization, cells should be fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 2% BSA before antibody staining . Co-staining experiments with markers for endoplasmic reticulum (ER) chaperones such as Calnexin and Calreticulin have demonstrated that EFHD2 does not significantly localize to the ER but rather resides in distinct perinuclear vesicular compartments . These findings can be further validated through subcellular fractionation techniques combining differential centrifugation with Western blot analysis using EFHD2 antibodies alongside compartment-specific markers . Advanced confocal microscopy with z-stack acquisition enables three-dimensional reconstruction of EFHD2 distribution patterns, while super-resolution techniques such as STED or STORM microscopy can provide nanoscale resolution of EFHD2 localization within specific cellular structures.

What approaches can detect EFHD2 interactions with tau proteins in neurodegenerative research?

To investigate EFHD2 interactions with tau proteins in neurodegenerative research, researchers can employ multiple complementary approaches including co-immunoprecipitation, proximity ligation assays, and fluorescence resonance energy transfer (FRET) techniques. Co-immunoprecipitation experiments using anti-EFHD2 antibodies can capture protein complexes containing both EFHD2 and tau from neural tissue or cell lysates, which can then be analyzed by Western blotting to confirm the interaction . For in situ detection of EFHD2-tau interactions, proximity ligation assays using primary antibodies against EFHD2 and tau can generate fluorescent signals only when the two proteins are in close proximity (<40 nm), enabling visualization of interaction sites within cellular contexts . FRET microscopy using fluorescently-labeled antibodies against EFHD2 and tau provides another approach to detect and quantify molecular interactions in living cells based on energy transfer between fluorophores when in close proximity. Recent findings have demonstrated that EFHD2 co-aggregates with both monomeric and filamentous tau in vitro, suggesting potential roles in tau pathology associated with neurodegenerative diseases . These protein interaction studies can be further validated using recombinant protein binding assays and structural analyses to characterize the molecular determinants governing EFHD2-tau associations.

How can EFHD2 antibodies be applied in tissue microarray analysis of neurodegenerative disorders?

EFHD2 antibodies can be strategically employed in tissue microarray (TMA) analysis to systematically investigate expression patterns and pathological associations across multiple neurodegenerative disorder samples. When preparing TMAs, researchers should include diverse brain regions (particularly those affected in tauopathies) from patients with various neurodegenerative conditions alongside age-matched controls . Immunohistochemical staining protocols should be optimized for sensitivity and specificity, typically involving antigen retrieval methods (heat-induced epitope retrieval in citrate buffer, pH 6.0), endogenous peroxidase blocking (0.3% H₂O₂), and appropriate blocking steps (5% normal serum) before applying HRP-conjugated anti-EFHD2 antibodies . For multiplex analysis, researchers can employ sequential immunostaining with antibodies against EFHD2 and disease-specific markers (such as phospho-tau, amyloid-β, or α-synuclein) using multispectral imaging systems to visualize co-localization patterns . Quantitative image analysis should measure parameters including EFHD2 expression intensity, distribution patterns (neuronal vs. glial), and co-localization coefficients with pathological markers using specialized software platforms. This comprehensive approach enables researchers to establish correlations between EFHD2 expression profiles and neuropathological features across different disease states, potentially revealing novel insights into disease mechanisms and progression patterns.

What are common false-positive signals when using EFHD2 antibodies and how can they be eliminated?

False-positive signals when using EFHD2 antibodies can arise from several sources including cross-reactivity with related proteins, non-specific binding to cellular components, and technical artifacts in the detection system. The structural similarity between EFHD2 and its homolog EFHD1 (69.7% sequence identity) presents a significant risk of cross-reactivity, although well-characterized monoclonal antibodies targeting the N-terminal region can effectively discriminate between these proteins . To minimize false-positive results, researchers should implement rigorous validation controls including parallel staining of EFHD2-knockout samples or EFHD2-silenced cells (such as WEHI231.shEFhd2), competitive blocking experiments with recombinant EFHD2 protein, and comparison with alternative EFHD2 antibody clones recognizing different epitopes . For immunohistochemistry or immunofluorescence applications, non-specific binding to cellular structures can be reduced by optimizing blocking protocols (using combinations of BSA, normal serum, and casein) and implementing more stringent washing steps with detergent-containing buffers . When using HRP-conjugated antibodies specifically, researchers should be mindful of potential background from endogenous peroxidase activity, which can be effectively quenched using hydrogen peroxide treatment prior to antibody application, as well as consider tyramide signal amplification methods for enhanced signal-to-noise ratios in challenging samples.

How should researchers interpret discrepancies between EFHD2 mRNA and protein expression data?

Discrepancies between EFHD2 mRNA and protein expression data require careful methodological consideration and biological interpretation. Such disparities may reflect post-transcriptional regulation mechanisms, differences in detection sensitivity, or technical limitations in either assay approach. Researchers should first verify technical aspects by ensuring that both mRNA quantification (via RT-qPCR or RNA-seq) and protein detection (via Western blot or flow cytometry) methods are properly validated using appropriate controls, reference genes, and calibration standards . Post-transcriptional regulatory mechanisms potentially affecting EFHD2 expression include microRNA-mediated repression, RNA-binding protein interactions, and variations in mRNA stability or translational efficiency, which can be investigated through targeted molecular approaches . Protein-level regulation through altered stability or proteolytic degradation may also contribute to observed discrepancies, as suggested by studies showing EFHD2 protein downregulation in rheumatoid arthritis patients' PBMCs through proteolytic processes . When encountering such discrepancies, researchers should consider employing complementary techniques such as polysome profiling to assess translational status of EFHD2 mRNA, pulse-chase experiments to determine protein half-life, or proteasome/lysosome inhibitors to investigate degradation pathways.

What are the critical controls for validating EFHD2 antibody specificity in immunological studies?

Rigorous validation of EFHD2 antibody specificity requires implementing a comprehensive suite of controls tailored to various experimental applications. The gold standard control involves parallel analysis of samples from EFHD2 knockout models or cells with confirmed EFHD2 silencing through shRNA/CRISPR approaches, which should show absence of signal when probed with specific anti-EFHD2 antibodies . For recombinant expression systems, comparison between untransfected cells, cells expressing EFHD2, and cells expressing the related EFHD1 protein provides critical specificity assessment . Competitive inhibition experiments using pre-incubation of the antibody with purified recombinant EFHD2 protein should abolish specific signals while pre-incubation with unrelated proteins (such as GST alone) should not affect staining intensity . When evaluating new antibody lots or working with challenging sample types, researchers should include positive controls from tissues or cell types known to express high EFHD2 levels (such as monocytes or neuronal tissues) alongside technical controls including isotype-matched irrelevant antibodies and secondary-only controls . For advanced applications like co-immunoprecipitation studies, reciprocal precipitation experiments (pulling down with anti-EFHD2 and probing for interacting partners, then reversing the approach) provide the most convincing evidence of specific protein interactions.

How can EFHD2 antibodies contribute to understanding neuroinflammatory processes?

EFHD2 antibodies offer powerful tools for investigating the protein's roles at the intersection of neuroinflammation and neurodegeneration through multi-parameter analysis of cellular phenotypes and protein interactions. Since EFHD2 is expressed in both immune cells and neuronal tissues, researchers can employ dual-labeling approaches with cell type-specific markers to track EFHD2 expression in microglia, astrocytes, and infiltrating immune cells within neuroinflammatory contexts . Flow cytometric analysis of brain immune cells using anti-EFHD2 antibodies combined with activation markers can reveal dynamic changes in EFHD2 expression during different phases of neuroinflammation . The significantly higher expression of EFHD2 in monocytes compared to other immune cells suggests potential functional importance in myeloid cell responses during neuroinflammatory processes, which can be explored through selective cell depletion and reconstitution experiments in combination with EFHD2 antibody-based detection methods . Researchers can further investigate EFHD2's role in the crosstalk between peripheral inflammation and central nervous system pathology by analyzing EFHD2 expression in peripheral blood mononuclear cells from patients with neurodegenerative disorders alongside markers of blood-brain barrier integrity and neuroinflammation . This integrated approach combining cellular phenotyping with protein interaction studies may reveal novel insights into how EFHD2 influences disease progression through modulation of inflammatory signaling pathways.

What methodological adaptations optimize EFHD2 detection in challenging tissue samples?

When working with challenging tissue samples, researchers can implement several methodological adaptations to enhance EFHD2 detection sensitivity and specificity. For formalin-fixed paraffin-embedded (FFPE) tissues with potential epitope masking, extended antigen retrieval methods combining heat and enzymatic approaches may be necessary, with optimization of buffer systems (citrate buffer pH 6.0 vs. Tris-EDTA pH 9.0) based on empirical testing . The use of signal amplification technologies such as tyramide signal amplification (TSA) can significantly enhance detection sensitivity for samples with low EFHD2 expression levels, enabling visualization of subtle expression patterns that might otherwise remain undetected . For highly autofluorescent tissues such as aged brain samples, researchers should incorporate additional treatments including Sudan Black B (0.1-0.3%) or commercial autofluorescence quenching reagents before immunostaining to improve signal-to-noise ratios . When working with frozen tissue sections that may exhibit high background, extended blocking protocols with combinations of different blocking agents (normal sera, BSA, casein, and commercial blocking solutions) can effectively reduce non-specific binding . For multiplex detection approaches, researchers should carefully select antibody combinations raised in different host species and optimize antibody concentration and incubation conditions for each marker individually before attempting simultaneous detection protocols.

How can researchers use EFHD2 antibodies to study protein degradation in disease models?

EFHD2 antibodies provide valuable tools for investigating protein degradation dynamics in disease models, particularly given evidence of proteolytic processing of EFHD2 in certain pathological conditions. To analyze EFHD2 degradation patterns, researchers should employ antibodies recognizing different epitopes (N-terminal and C-terminal regions) in parallel Western blot analyses, which can reveal specific fragmentation patterns or post-translational modifications . When investigating potential proteolytic mechanisms, pharmacological inhibitors targeting different proteolytic pathways (proteasomal, lysosomal, and specific proteases) can be applied to cells or tissues before EFHD2 detection to identify the responsible degradation systems . Pulse-chase experiments combined with immunoprecipitation using anti-EFHD2 antibodies enable measurement of protein half-life under normal and pathological conditions, while cycloheximide-chase assays can distinguish between reduced synthesis and enhanced degradation as causes for decreased EFHD2 levels . For in vivo studies, ubiquitin-proteasome system reporters combined with EFHD2 antibody staining can reveal spatial and temporal relationships between proteolytic activity and EFHD2 expression changes. These methodological approaches are particularly relevant for rheumatoid arthritis research, where EFHD2 has been reported to be downregulated in peripheral blood mononuclear cells through proteolytic processes, and for neurodegenerative disorders, where aberrant protein degradation pathways intersect with tau pathology that may involve EFHD2 .