EFHD2 Antibody, FITC conjugated

What is EFHD2 and what cellular functions does it perform?

EFHD2 (also known as Swiprosin-1) is a highly conserved calcium-binding adaptor protein belonging to the EF hand family. It functions in calcium signaling pathways in both immune and neuronal cells. EFHD2 has a close homologue called EFHD1 (Swiprosin-2) with approximately 69.7% sequence identity . EFHD2 is abundantly expressed in brain tissue and immune cells, particularly B cells and monocytes . It has been implicated in B cell receptor signaling and various neuropathological disorders . The protein contains several functional domains: an N-terminal low complexity region, a proline-rich stretch, two EF hands, and a C-terminal coiled-coil domain, each contributing to its cellular functions .

What is the subcellular localization of EFHD2 in immune cells?

Immunofluorescence studies using specific anti-EFHD2 monoclonal antibodies have revealed two major pools of EFHD2 in B cells: one at the plasma membrane and another in intracellular compartments . The intracellular pool does not co-localize with endoplasmic reticulum markers (Calnexin and Calreticulin), suggesting EFHD2 resides in different vesicular structures . Additionally, subcellular fractionation assays have confirmed that EFHD2 does not localize to the nucleus but is found in membrane and perinuclear vesicular structures . This distribution pattern suggests potential roles for EFHD2 in membrane-associated signaling and vesicular trafficking in immune cells.

How can I verify the specificity of an anti-EFHD2 antibody before experimentation?

The specificity of anti-EFHD2 antibodies should be rigorously validated through multiple approaches:

• Comparative cell models: Test the antibody on cell lines with confirmed presence or absence of EFHD2 expression. For example, using WEHI231 B cells with EFHD2 knockdown versus cells with reconstituted EFHD2 expression provides an excellent control system .

• Cross-reactivity testing: Evaluate whether the antibody recognizes related proteins, particularly EFHD1. Western blotting with lysates containing overexpressed EFHD1 and EFHD2 can confirm specificity .

• Blocking experiments: Pre-incubate the anti-EFHD2 antibody with purified EFHD2 protein (e.g., GST-EFHD2) before staining, which should abolish specific binding if the antibody is truly EFHD2-specific .

• Multiple detection methods: Confirm specificity across different applications (Western blotting, immunofluorescence, flow cytometry, immunoprecipitation) as antibody performance can vary between techniques .

What methodological considerations are important when using FITC-conjugated EFHD2 antibodies in flow cytometry?

When using FITC-conjugated EFHD2 antibodies for flow cytometry, researchers should consider:

• Fixation and permeabilization optimization: The research indicates that PFA fixation with 0.1% Tween-20 permeabilization at 37°C provides optimal results for intracellular EFHD2 detection .

• Antibody titration: Perform dose-dependent staining to determine the optimal antibody concentration that provides specific signal without excessive background. Flow cytometric analysis can quantify EFHD2 expression in a linear manner over at least two log scales .

• Appropriate controls: Include:

  • Isotype-matched control antibodies (e.g., IgG1κ for mouse monoclonal antibodies)

  • Blocking controls (pre-incubation with recombinant EFHD2)

  • Positive and negative cell populations (cells known to express high or low levels of EFHD2)

• Gating strategy: Clearly define the populations of interest (e.g., monocytes versus B cells) when analyzing heterogeneous samples like PBMCs .

What fixation and permeabilization methods are most effective for different EFHD2 detection techniques?

The effectiveness of different fixation and permeabilization methods varies depending on the detection technique, as summarized in the following table:

For flow cytometry applications, PFA fixation followed by permeabilization with 0.1% Tween-20 at 37°C is particularly effective for intracellular EFHD2 detection . When working with tissue sections, anti-EFHD2 MAbs have been successfully used on both paraffin-embedded tissues (with antigen retrieval) and cryosections .

How can I design experiments to study EFHD2 expression patterns across different immune cell populations?

To comprehensively analyze EFHD2 expression patterns across immune cell populations:

• Multi-parameter flow cytometry: Combine FITC-conjugated EFHD2 antibody with markers for specific immune cell subsets (CD19 for B cells, CD14 for monocytes, CD3 for T cells, etc.) to simultaneously assess EFHD2 expression across multiple populations.

• Quantitative analysis: Establish a standardized protocol for relative quantification. Studies have shown that human monocytes express approximately five times more EFHD2 than human B cells .

• Population isolation: When practical, use cell sorting or magnetic isolation to purify specific cell populations before analysis to minimize interference from other cell types.

• Developmental analysis: For B cell-focused studies, compare EFHD2 expression across differentiation stages using established cell lines representing pro-B (38B9), pre-B (NFS5), activated immature and mature B cells (WEHI231, CH27.LX), and plasma cells (Ag8) .

What techniques can be used to simultaneously assess EFHD2 localization and expression levels?

Several complementary approaches can be utilized:

• Confocal microscopy with quantitative image analysis: Allows visualization of subcellular localization while enabling quantification of fluorescence intensity as a measure of expression levels.

• Subcellular fractionation followed by Western blotting: Provides biochemical confirmation of EFHD2 distribution between membrane, cytosolic, nuclear, and vesicular compartments, with quantitative assessment of relative abundance in each fraction .

• Combined flow cytometry and imaging cytometry: Modern imaging flow cytometers can simultaneously quantify total EFHD2 levels and assess its subcellular distribution patterns in large cell populations.

• Proximity ligation assays: Can be used to investigate EFHD2 interactions with other proteins in specific subcellular compartments, providing functional context to localization data.

How can EFHD2 antibodies be utilized to investigate potential links between EFHD2 and neurodegenerative disorders?

EFHD2 has been implicated in several neurodegenerative conditions, and researchers can investigate these connections using FITC-conjugated EFHD2 antibodies through:

• Analysis of peripheral immune cells as biomarkers: PBMC have been used to assess transcriptional differences and alterations in proteolytic pathways between healthy donors and patients with Alzheimer's and Parkinson's disease . Quantifying EFHD2 expression and degradation patterns in PBMCs from patients versus controls could identify potential disease biomarkers.

• Assessment of proteolytic degradation: Combining N-terminal binding antibodies (like the described monoclonal antibodies) with commercially available C-terminal binding antibodies can help define EFHD2 cleavage patterns that may be altered in disease states .

• Comparative analysis across disorders: EFHD2 has been linked to behavioral brain disorders such as schizophrenia and neurodegenerative disorders . Systematic comparison of EFHD2 expression, localization, and processing across these conditions could reveal disorder-specific patterns.

• Correlation with inflammatory markers: Since EFHD2 has been shown to be down-regulated in PBMCs of rheumatoid arthritis patients through proteolytic processes , similar mechanisms might be active in neuroinflammatory components of neurodegenerative disorders.

What experimental approaches can detect potential post-translational modifications or proteolytic processing of EFHD2?

Several techniques can reveal post-translational modifications and proteolytic processing:

• Western blot analysis with multiple antibodies: Using antibodies that recognize different epitopes (N-terminal and C-terminal) can reveal specific degradation products. The presence of differentially migrating bands on Western blots may indicate post-translational modifications or degradation products .

• Immunoprecipitation followed by mass spectrometry: This can identify specific post-translational modifications and cleavage sites with high precision.

• 2D gel electrophoresis: This approach can separate EFHD2 variants with subtle differences in charge or size resulting from post-translational modifications.

• Pulse-chase experiments: These can determine the half-life of EFHD2 and track proteolytic processing in various cell types or disease models.

• Inhibitor studies: Using specific protease inhibitors can help identify the enzymes responsible for EFHD2 processing that might be dysregulated in disease states.

How do calcium levels affect EFHD2 detection with antibodies, and what controls should be implemented?

As EFHD2 is a calcium-binding protein with two EF hand domains, calcium levels may affect its conformational state and potentially antibody recognition:

• Calcium chelation experiments: Compare EFHD2 detection in the presence versus absence of calcium chelators (EGTA, EDTA) to determine if antibody binding is calcium-dependent.

• Calcium concentration gradients: Assess whether varying calcium concentrations in buffers impact detection efficiency or subcellular localization patterns.

• Calcium ionophore treatments: Pre-treatment of cells with calcium ionophores before fixation can reveal whether calcium influx alters EFHD2 distribution or antibody accessibility.

• EF hand mutants as controls: Cells expressing EFHD2 with mutations in the EF hand domains (ΔEF1, ΔEF2) provide excellent controls to determine whether calcium binding affects antibody recognition.

What could explain variability in EFHD2 detection between experiments or cell types?

Variability in EFHD2 detection may arise from several factors:

• Differential expression levels: As demonstrated by the ~5-fold higher expression in monocytes versus B cells, cell type-specific expression levels vary significantly .

• Subcellular localization differences: The distribution between membrane and intracellular pools may vary between cell types or activation states, affecting accessibility for antibody binding .

• Post-translational modifications: These may mask epitopes or alter protein conformation in different cellular contexts.

• Fixation and permeabilization effects: Different protocols may differentially preserve EFHD2 or affect epitope accessibility, as indicated by the varying effectiveness of different methods .

• Antibody clone specificities: The four monoclonal antibodies described (A4.15.28, A4.15.48, A4.18.18, E7.20.23) may have subtle differences in epitope recognition within the N-terminal region, which could affect detection in different experimental contexts .

How should researchers interpret multiple bands in Western blot analysis of EFHD2?

When multiple bands appear in Western blots probed with anti-EFHD2 antibodies:

• Expected main band: The primary band should correspond to the predicted molecular weight of full-length EFHD2.

• Higher molecular weight bands: May represent:

  • Post-translational modifications (phosphorylation, ubiquitination, etc.)

  • Dimerization or complex formation with other proteins that resist denaturation

• Lower molecular weight bands: May indicate:

  • Proteolytic degradation products, potentially biologically relevant in disease states

  • Alternative splicing variants

  • Non-specific antibody binding (rule out with appropriate controls)

• Verification approaches:

  • Compare patterns between different anti-EFHD2 antibodies recognizing distinct epitopes

  • Use lysates from EFHD2-deficient cells as negative controls

  • For recombinant expression systems, compare to endogenous expression patterns

The research notes that when expressing EFHD2 from cDNA constructs, differentially migrating bands were observed that represented transfected protein, potentially indicating post-translational modifications or degradation products of the overexpressed protein .

What considerations are important when normalizing EFHD2 expression data across different experimental conditions?

Proper normalization of EFHD2 expression data requires:

• Consistent loading controls: For Western blot, use housekeeping proteins (β-actin, GAPDH) or total protein staining methods (Ponceau S, Coomassie).

• Flow cytometry standardization:

  • Use calibration beads with defined fluorescence intensities

  • Include a common reference cell population across experiments

  • Report data as molecules of equivalent soluble fluorochrome (MESF) rather than arbitrary units

• Cell type-specific baselines: Given the significant difference in EFHD2 expression between cell types (e.g., monocytes vs. B cells) , establish baseline expression for each cell type rather than using a single reference point.

• Paired experimental designs: When possible, process and analyze samples from different conditions simultaneously to minimize technical variability.

• Multiple detection methods: Validate key findings using complementary techniques (flow cytometry, Western blot, qPCR) to ensure observations are not method-dependent.

How might EFHD2 antibodies be utilized to investigate potential biomarkers in inflammatory or neurodegenerative conditions?

EFHD2 antibodies, particularly FITC-conjugated versions for flow cytometry, offer promising applications for biomarker studies:

• Longitudinal patient monitoring: Standardized flow cytometry protocols could track EFHD2 expression in PBMCs from patients with neurological disorders over time, potentially correlating changes with disease progression .

• Differential diagnosis: Compare EFHD2 expression patterns across different neurodegenerative diseases to identify disease-specific signatures.

• Treatment response markers: Investigate whether therapeutic interventions normalize aberrant EFHD2 expression or processing patterns in patient cells.

• Correlation with established biomarkers: Combine EFHD2 analysis with established biomarkers (e.g., Aβ, tau, inflammatory cytokines) to develop multiparameter diagnostic panels.

• Proteolytic patterns: As EFhd2 has been shown to be downregulated through proteolytic processes in rheumatoid arthritis, similar mechanisms might apply in neuroinflammatory conditions .

What approaches can be used to discriminate between closely related proteins when using immunological detection methods?

To ensure specificity when detecting EFHD2 in the presence of related proteins like EFHD1:

• Epitope selection: Target regions of highest sequence divergence, such as the N-terminal portion where EFHD2 and EFHD1 differ most significantly .

• Validation in knockout/knockdown systems: Test antibodies in systems where the target protein expression is selectively eliminated through genetic approaches (as demonstrated with WEHI231.shEFhd2 cells) .

• Cross-absorption of antibodies: Pre-incubate antibodies with recombinant related proteins to remove cross-reactive antibodies.

• Competitive binding assays: Use unlabeled competing antibodies to confirm binding site specificity.

• Parallel detection strategies: Combine antibody-based detection with orthogonal methods like mass spectrometry to validate protein identity.

The described anti-EFHD2 monoclonal antibodies demonstrate excellent discrimination between EFHD2 and EFHD1 due to their specificity for the N-terminal region, providing a model for developing highly specific antibodies against related protein family members .