EFCAB7 Antibody

What is EFCAB7 and what is its functional role in cellular signaling?

EFCAB7 (EF-hand calcium binding domain 7) is a protein that forms part of the EvC complex, which positively regulates the Hedgehog (Hh) signaling pathway. The protein contains at least 8 EF-hand domains arranged in two distinct runs: an N-terminal set of 5 EF-Hands (EF1–5), followed by an uncharacterized globular domain, followed by a second run of at least 3 EF-Hands (EF6–8) followed by another globular domain .

EFCAB7 functions as an adaptor protein that links the EVC-EVC2 complex to IQCE. It uses its ECH2 domain to engage the W-peptide in EVC2 and its EF1-5 domains to bind to IQ domains in IQCE . This complex formation is critical for:

• Anchoring the EVC-EVC2 complex in a signaling microdomain at the base of cilia

• Proper localization of the complex in the EvC zone

• Enabling ciliary Hedgehog signaling transduction

• Activating the transcription factor GLI2

Mutations in EFCAB7 can disrupt this complex formation, leading to impaired Hedgehog signaling similar to what is observed in Ellis van Creveld and Weyers syndromes .

What applications are EFCAB7 antibodies validated for?

Based on multiple commercial sources, EFCAB7 antibodies have been validated for several experimental applications:

Many antibodies are specifically validated against human EFCAB7, with some showing cross-reactivity with mouse samples. When selecting an antibody, researchers should verify the specific validation data for their intended application and species .

Where is EFCAB7 predominantly localized in cells?

EFCAB7 is predominantly localized at the base of primary cilia, specifically in a region called the EvC zone. Immunofluorescence studies using both endogenous and YFP-tagged EFCAB7 have confirmed this localization pattern .

In human retinal pigment epithelial (RPE1) cells, endogenous EFCAB7 protein has been observed at the basal body region of primary cilia . This specific localization is crucial for its function in Hedgehog signaling, as it helps position the EvC complex correctly within the ciliary architecture.

The localization pattern is consistent with EFCAB7's role as an adaptor protein that tethers the EVC-EVC2 complex to the ciliary base, creating a signaling microdomain essential for proper Hedgehog signal transduction .

How can I optimize antibody-based detection of EFCAB7 in ciliary structures?

Detecting EFCAB7 in ciliary structures requires careful optimization due to its specific localization in the EvC zone at the base of cilia. Based on published research protocols:

• Cell Preparation:

  • Seed approximately 0.6 × 10^5 cells per well onto Lab-Tek chambered slides

  • Culture for 24 hours before transfection or treatment

  • For visualizing ciliary proteins, starve cells in serum-free DMEM for 24 hours

• Fixation and Staining Protocol:

  • Fix cells in 4% paraformaldehyde for 15 minutes at 4°C

  • Use dual immunostaining with ciliary markers to accurately identify EFCAB7 localization:

    • Mouse anti-Acetylated tubulin (1:1000 dilution) or mouse anti-γ-tubulin (1:200 dilution) for ciliary structures

    • Rabbit anti-EFCAB7 (1:100 dilution) for target protein

• Co-localization Analysis:

  • Use confocal microscopy with z-stack imaging to precisely determine EFCAB7 localization relative to the ciliary base

  • Include co-staining with other EvC complex components (EVC, EVC2, IQCE) for comprehensive analysis

• Controls:

  • Include EFCAB7 knockout cells as negative controls to confirm antibody specificity

  • Consider using YFP-tagged EFCAB7 expression as a positive control

This approach has successfully demonstrated EFCAB7 localization at the base of cilia in multiple cell types and experimental conditions.

What are the critical considerations when using EFCAB7 antibodies to study Hedgehog signaling pathways?

When studying Hedgehog signaling using EFCAB7 antibodies, researchers should consider several critical factors:

These considerations will help ensure that experimental observations reflect genuine roles of EFCAB7 in Hedgehog signaling rather than technical artifacts.

How do mutations in EFCAB7 affect antibody binding and experimental interpretation?

Mutations in EFCAB7 can significantly impact antibody recognition and experimental interpretations, particularly when studying disease-associated variants:

• Splicing Variant Effects:

  • The splicing variant c.683-1G>C causes exon 6 skipping, resulting in a truncated protein

  • This 122 bp deletion alters the protein's tertiary structure, potentially affecting epitope accessibility

  • Antibodies targeting regions encoded by or adjacent to exon 6 may show reduced binding to this variant

• Domain-Specific Antibody Selection:

  • For detecting all potential EFCAB7 variants, choose antibodies targeting conserved regions outside mutation hotspots

  • Antibodies targeting the N-terminal region (AA 1-158) may detect most variants including truncated forms

  • C-terminal antibodies might fail to detect truncated proteins

• Protein Stability Considerations:

  • Mutations like the exon 6 deletion significantly reduce protein stability

  • This leads to weaker signal intensity that might be misinterpreted as reduced expression

  • Consider using proteasome inhibitors (MG132) in experimental designs when studying unstable variants

• Subcellular Localization Analysis:

  • Some mutations may not affect ciliary localization despite impairing function

  • The c.683-1G>C variant protein still localizes to the basal body of primary cilia similarly to wild-type EFCAB7

  • Careful co-localization studies with proper controls are essential for accurate interpretation

When designing experiments to study EFCAB7 variants, researchers should employ multiple antibodies targeting different epitopes and combine protein detection with functional assays to ensure comprehensive analysis.

What strategies can resolve contradictory findings when using different EFCAB7 antibodies?

Researchers often encounter contradictory results when using different antibodies against the same target. For EFCAB7, the following strategies can help resolve such discrepancies:

• Knockout-Based Validation:

  • Generate EFCAB7 knockout cells via CRISPR/Cas9 as definitive negative controls

  • Test all antibodies against both wild-type and knockout samples in parallel

  • Any signal persisting in knockout samples indicates non-specific binding

• Epitope Mapping Analysis:

  • Determine which protein domain each antibody targets

  • EFCAB7 contains multiple EF-hand domains and other structural features that may affect epitope accessibility

  • Create domain deletion constructs to identify the specific binding regions for each antibody

• Application-Specific Optimization:

  • Recognition in one application doesn't guarantee success in others

  • For western blotting: optimize denaturation conditions that may expose different epitopes

  • For immunohistochemistry: test multiple antigen retrieval methods

  • For immunofluorescence: compare different fixation protocols (PFA vs. methanol)

• Orthogonal Validation Approaches:

  • Complement antibody-based detection with genetic approaches:

    • Express tagged versions of EFCAB7 (YFP/GFP-EFCAB7)

    • Perform siRNA knockdown followed by rescue with siRNA-resistant constructs

    • Use mass spectrometry to confirm protein identity in immunoprecipitates

• Standardized Testing Protocol:

  • Adopt the Antibody Characterization through Open Science (YCharOS) approach:

    • Side-by-side comparison of all antibodies against a target

    • Consistent protocols across all antibodies

    • Technical peer review of characterization data

How can EFCAB7 antibodies be used to investigate congenital heart defects?

Recent research has identified EFCAB7 variants in patients with Tetralogy of Fallot (TOF), suggesting its role in congenital heart development. Researchers investigating this connection can utilize EFCAB7 antibodies through the following approaches:

• Patient Sample Analysis:

  • Perform immunohistochemistry on cardiac tissues from patients with TOF or other congenital heart defects

  • Compare EFCAB7 expression and localization patterns between affected and control tissues

  • Use antibodies validated for IHC-P at dilutions of 1:50-1:200

• Animal Model Validation:

  • Utilize EFCAB7 antibodies to characterize expression in heart tissues from knock-in mice carrying patient-specific variants

  • Analyze protein expression at different developmental stages (E10.5-P1) focusing on the cardiac outflow tract

  • Compare with cilia markers to assess ciliogenesis in cardiac tissues

• Mechanistic Investigation:

  • Monitor downstream Hedgehog pathway components in cardiac development:

    • Gli transcription factor activation and nuclear localization

    • Expression of Gli target genes important for heart development (Myh6, Zfpm1, Nkx2-5)

    • Smoothened (SMO) localization to primary cilia in cardiac progenitor cells

• Cellular Phenotype Characterization:

  • Use EFCAB7 antibodies in conjunction with CUT&Tag technology to analyze Gli binding to target genes

  • Perform immunofluorescence to assess cilia number and length in cardiac cells

  • Combine with RNA-seq data to correlate EFCAB7 function with gene expression changes during heart development

This integrated approach can help establish the causal relationship between EFCAB7 dysfunction and congenital heart defects, potentially revealing new diagnostic or therapeutic targets.

What are the best practices for validating new EFCAB7 antibodies for research use?

Validating new EFCAB7 antibodies requires a comprehensive approach that ensures specificity, sensitivity, and reproducibility across applications. Based on recent advances in antibody validation methodologies:

• Genetic Strategy Implementation:

  • Generate EFCAB7 knockout cell lines using CRISPR/Cas9

  • For essential genes, use knockdown approaches with multiple siRNAs

  • Test antibodies on both wild-type and knockout/knockdown samples in parallel

• Multi-Application Testing:

  • Validate each antibody in all intended applications (WB, IP, IF, IHC)

  • For western blotting: confirm band at expected molecular weight (72 kDa)

  • For immunofluorescence: verify co-localization with ciliary markers

  • For immunoprecipitation: confirm enrichment by mass spectrometry

• Cross-Reactivity Assessment:

  • Test on protein arrays containing EFCAB7 and unrelated proteins

  • Some commercial antibodies are verified against arrays containing 383+ non-specific proteins

  • Evaluate cross-reactivity with related EF-hand domain-containing proteins

• Epitope Mapping and Characterization:

  • Determine the specific region recognized by the antibody

  • Create a panel of domain deletion constructs to map binding sites

  • Test antibodies against recombinant protein fragments corresponding to different domains

• Reproducibility Evaluation:

  • Test across multiple lots of the same antibody

  • Verify performance in different cell types and tissues

  • Document optimal working conditions (dilutions, incubation times, buffers)

• Documentation and Data Sharing:

  • Follow the YCharOS model of comprehensive reporting

  • Document all validation data in standardized formats

  • Share validation data through open repositories like ZENODO

This rigorous validation approach ensures that antibodies used in EFCAB7 research produce reliable and reproducible results, advancing our understanding of this protein's function in health and disease.

How can EFCAB7 antibodies be used to study protein-protein interactions within the EvC complex?

EFCAB7 forms a tetrameric complex with IQCE, EVC, and EVC2, known as the EvC complex. Researchers investigating these interactions can leverage EFCAB7 antibodies through several specialized approaches:

• Co-Immunoprecipitation (Co-IP) Strategies:

  • Use EFCAB7 antibodies for pull-down experiments to capture native EvC complexes

  • Optimize lysis conditions to preserve protein-protein interactions:

    • Use non-denaturing detergents like NP-40 or digitonin

    • Include protease inhibitors and phosphatase inhibitors

    • Maintain physiological salt concentrations

  • Analyze immunoprecipitates by western blotting for EVC, EVC2, and IQCE

• Interaction Domain Mapping:

  • Combine EFCAB7 antibodies with domain-specific mutant constructs:

    • ECH2 domain mutants to disrupt interaction with the W-peptide of EVC2

    • EF1-5 domain mutants to disrupt interaction with IQ domains of IQCE

  • Use truncation mutants like ΔIQ-IQCE (1-552) that fail to interact with EFCAB7

• Proximity Ligation Assays (PLA):

  • Use EFCAB7 antibodies in conjunction with antibodies against other complex components

  • PLA provides spatial resolution of protein interactions within intact cells

  • Quantify interaction signals at the base of cilia versus other cellular compartments

• Stimulus-Dependent Interaction Analysis:

  • Investigate complex formation under different signaling conditions:

    • Before and after Hedgehog pathway stimulation with SAG

    • During ciliogenesis following serum starvation

    • In response to calcium flux (relevant for EF-hand domains)

• Reconstitution Systems:

  • Use HEK 293T cells for complex reconstitution experiments

  • Co-express YFP-tagged complex components with untagged partners

  • Iteratively move the YFP tag between complex members to confirm tetrameric assembly

  • Visualize complex formation by Coomassie staining or immunoblotting

These approaches can reveal the structural and functional organization of the EvC complex, providing insights into how EFCAB7 coordinates its assembly and localization at the base of primary cilia.

What are common troubleshooting strategies for weak or nonspecific EFCAB7 antibody signals?

Researchers working with EFCAB7 antibodies may encounter signal issues that require specific troubleshooting approaches:

• Weak Signal Troubleshooting:

  • Protein Expression Level Factors:

    • EFCAB7 undergoes rapid turnover in cells

    • Consider using proteasome inhibitors (MG132) to prevent degradation

    • Verify expression in your cell type using transcript data from resources like the Human Protein Atlas

  • Technical Optimization:

    • Increase antibody concentration (try 1:50 for IHC if 1:200 is weak)

    • Extend primary antibody incubation time (overnight at 4°C)

    • Optimize antigen retrieval for fixed tissues (try citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

    • Use signal amplification systems (HRP-polymer or TSA)

• Nonspecific Signal Remediation:

  • Blocking Optimization:

    • Test different blocking agents (BSA, normal serum, commercial blockers)

    • Extend blocking time to reduce background (2 hours at room temperature)

    • Include 0.1-0.3% Triton X-100 in blocking buffer for better penetration

  • Antibody Specificity Enhancement:

    • Pre-adsorb antibody with cell lysates from EFCAB7 knockout cells

    • Perform peptide competition assays using the immunogen peptide

    • Use more stringent washing conditions (higher salt concentration)

• Application-Specific Approaches:

  • For Western Blotting:

    • Optimize transfer conditions for high molecular weight proteins

    • Use freshly prepared samples to avoid degradation

    • Try different membrane types (PVDF vs. nitrocellulose)

    • Load higher protein amounts (50-100 μg) when detecting endogenous EFCAB7

  • For Immunofluorescence:

    • Compare different fixation methods (4% PFA, methanol, or combination)

    • Optimize permeabilization conditions

    • Use confocal microscopy to improve signal-to-noise ratio

    • Consider antifade mounting media to preserve fluorescence

• Storage and Handling:

  • Store antibodies according to manufacturer recommendations (typically aliquoted at -20°C)

  • Avoid freeze-thaw cycles that can degrade antibody quality

  • For long-term storage, use glycerol-containing buffers as recommended

Implementing these troubleshooting strategies systematically can help researchers overcome technical challenges and obtain reliable results with EFCAB7 antibodies.

How can researchers optimize protocols for detecting EFCAB7 in different tissue types?

Detecting EFCAB7 across diverse tissue types requires optimization strategies tailored to the specific characteristics of each tissue:

• Tissue-Specific Fixation Protocols:

  • For Brain Tissues:

    • Use shorter fixation times (12-24 hours) with 4% PFA

    • Consider transcardial perfusion for animal samples

    • EFCAB7 expression data from Allen Brain Atlas can guide expected detection patterns

  • For Heart Tissues:

    • For embryonic hearts: fix in 4% PFA for 24 hours at 4°C

    • For adult hearts: use shorter fixation (6-12 hours) to prevent overfixation

    • Consider sectioning before fixation for larger specimens

  • For High-Fat Tissues:

    • Extend fixation time slightly to ensure penetration

    • Include additional washing steps to remove lipids

    • Consider lipid removal agents if background is problematic

• Antigen Retrieval Optimization:

  • Heat-Induced Epitope Retrieval (HIER):

    • For formalin-fixed tissues: citrate buffer (pH 6.0) at 95-100°C for 20 minutes

    • For difficult tissues: try Tris-EDTA (pH 9.0) as an alternative

    • Optimize pressure and temperature based on tissue density

  • Enzymatic Retrieval:

    • For fibrotic tissues: proteinase K treatment (10-20 μg/ml for 10-15 minutes)

    • For heavily cross-linked samples: trypsin digestion (0.05% for 5-15 minutes)

    • Always include a non-treated control section

• Detection System Selection:

  • For Low Expression Tissues:

    • Use amplification systems like tyramide signal amplification (TSA)

    • Consider polymer-based detection systems for enhanced sensitivity

    • Extend chromogen development time with careful monitoring

  • For Autofluorescent Tissues:

    • Implement autofluorescence quenching steps (Sudan Black B, TrueBlack)

    • Select fluorophores with emission spectra distinct from autofluorescence

    • Use spectral imaging to separate specific signal from autofluorescence

• Tissue-Specific Positive Controls:

  • Include known EFCAB7-expressing tissues as positive controls

  • For human tissues: kidney shows reliable expression

  • For mouse tissues: skeletal muscle shows detectable levels

• Counterstaining Strategies:

  • Use nuclear counterstains to provide context (DAPI, hematoxylin)

  • Consider co-staining with cell type-specific markers to identify EFCAB7-expressing cells

  • Include ciliary markers (acetylated tubulin, γ-tubulin) to confirm EvC zone localization

By systematically optimizing these parameters for each tissue type, researchers can achieve consistent and specific detection of EFCAB7 across diverse experimental samples.

How are EFCAB7 antibodies being used to investigate novel roles in developmental disorders?

Recent research has begun to uncover EFCAB7's involvement in developmental disorders beyond its established role in Hedgehog signaling. Researchers are using EFCAB7 antibodies to explore these emerging connections:

• Congenital Heart Defects:

  • EFCAB7 splicing variants have been linked to Tetralogy of Fallot (TOF)

  • Researchers are using antibodies to track EFCAB7 expression during cardiac outflow tract development

  • Immunostaining of embryonic hearts from knockout mice reveals ventricular septal defects, atrial septal defects, and narrowed pulmonary arteries

• Ciliopathy-Related Phenotypes:

  • EFCAB7 mutations have been identified in patients with ciliopathy features

  • Antibodies are being used to assess cilia number, length, and morphology in patient-derived cells

  • Researchers are investigating EFCAB7's role in ciliary transport mechanisms beyond Hedgehog signaling

• Transcriptional Regulation Studies:

  • Integration of CUT&Tag data with EFCAB7 antibodies is revealing roles in Gli transcription factor binding

  • This approach has identified downstream targets including cardiac development genes (Myh6, Zfpm1, Nkx2-5)

  • EFCAB7 antibodies are being used to track protein localization during transcriptional complex assembly

• Calcium Signaling Integration:

  • The EF-hand domains in EFCAB7 suggest potential calcium-dependent functions

  • Researchers are using antibodies to investigate relationships between calcium flux and EFCAB7 localization/function

  • This may reveal novel signaling pathways independent of its established role in ciliary Hedgehog signaling

• Evolutionary Studies:

  • EFCAB7 appears to have been important for adaptation of cilia for animal-specific signaling networks

  • Antibodies are being used to compare EFCAB7 localization and function across evolutionary diverse organisms

  • This evolutionary perspective may reveal fundamental principles of ciliary specialization

These emerging research directions highlight how EFCAB7 antibodies are enabling discoveries beyond the protein's initially characterized functions, potentially revealing new therapeutic targets for developmental disorders.

What are the latest methodological advances in using EFCAB7 antibodies for high-resolution imaging?

Recent technological advances have enhanced the capabilities of EFCAB7 antibodies for high-resolution imaging of ciliary structures and protein complexes:

• Super-Resolution Microscopy Applications:

  • STORM and PALM Techniques:

    • Single-molecule localization microscopy enables ~20nm resolution of EFCAB7 at ciliary base

    • Use of photoswitchable fluorophore-conjugated secondary antibodies enhances localization precision

    • This approach has revealed distinct subdomains within the EvC zone

  • SIM and Airyscan Approaches:

    • Structured illumination microscopy provides 2x resolution improvement without special fluorophores

    • Particularly useful for live-cell imaging of EFCAB7-GFP dynamics

    • Airyscan detection improves signal-to-noise ratio for weak EFCAB7 signals

• Cryo-Electron Microscopy Integration:

  • Correlative Light and Electron Microscopy (CLEM):

    • EFCAB7 antibodies with gold nanoparticle labels enable precise ultrastructural localization

    • Correlative approaches link fluorescence patterns to electron-dense structures

    • Reveals how EFCAB7 organizes molecular architecture at the ciliary base

• Expansion Microscopy:

  • Physical expansion of hydrogel-embedded samples increases effective resolution

  • Compatible with standard EFCAB7 antibodies and conventional microscopes

  • Particularly valuable for resolving the spatial relationship between EFCAB7 and other EvC complex components

• Multiplexed Antibody Imaging:

  • Iterative Staining Methods:

    • Sequential staining and elution allows visualization of 20+ proteins in the same sample

    • Enables comprehensive mapping of EFCAB7 interactions within intact cilia

  • Mass Cytometry Imaging:

    • Metal-conjugated antibodies and mass spectrometry detection overcome fluorescence limitations

    • Allows simultaneous detection of EFCAB7 with numerous other proteins without spectral overlap

• Live-Cell Imaging Strategies:

  • Nanobody-Based Detection:

    • Single-domain antibody fragments enable live-cell visualization of EFCAB7

    • Smaller size improves penetration and reduces interference with protein function

  • CRISPR Knock-in Tags:

    • Endogenous tagging of EFCAB7 eliminates overexpression artifacts

    • Enables physiological visualization of dynamics during ciliogenesis and signaling

These methodological advances are providing unprecedented insights into EFCAB7's spatial organization, dynamics, and functional interactions within the ciliary signaling machinery.