efcab11 Antibody

What is EFCAB11 and why is it of interest to researchers?

EFCAB11 is a protein-coding gene located on chromosome 14q32.11 that encodes an EF-hand domain-containing protein predicted to enable calcium ion binding activity . It has drawn scientific interest due to its association with several conditions, including:

• Genetic variations at loci involved in immune responses that are risk factors for hepatocellular carcinoma

• Genome-wide association with adverse metabolic responses to hydrochlorothiazide (HCTZ) in African Americans

• Involvement in monoamine metabolite levels in human cerebrospinal fluid

Additionally, in rat models, EFCAB11 has been implicated in microtubule cytoskeleton organization processes , suggesting potential roles in cellular structure and function.

What types of EFCAB11 antibodies are available for research applications?

Multiple types of EFCAB11 antibodies are available for research purposes:

• Polyclonal antibodies: Typically raised in rabbits against specific amino acid sequences of human EFCAB11 (such as AA 1-163 or the sequence MFFSEARARSRTWEASPSEHRKWVEVFKACDEDHKGYLSREDFKTAVVMLFGYKPSKIEVDSVMSS)

• Monoclonal antibodies: Available from mouse hosts (e.g., clone 3B3)

• Application-specific antibodies: Optimized for techniques including Western blotting, ELISA, immunocytochemistry, and immunofluorescence

When selecting an antibody, researchers should consider the specific experimental application, species reactivity (most are human-specific), and whether a monoclonal or polyclonal approach better suits their research needs.

What is the relationship between EFCAB11 and TDP1?

EFCAB11 and TDP1 (Tyrosyl-DNA phosphodiesterase 1) form a bidirectional gene pair, with transcription start sites that are neighboring but directed away from each other . This genomic arrangement has important regulatory implications:

• Both genes can be simultaneously silenced by hypermethylation of CpG islands in their shared bidirectional promoter

• In the HOP_62 cell line, both EFCAB11 and TDP1 are selectively downregulated due to this epigenetic mechanism

• This relationship may have significance in cancer biology, as TDP1 plays a role in DNA repair processes, and its inactivation may influence cellular responses to topoisomerase 1-targeted drugs

This genomic organization suggests that EFCAB11 expression patterns may serve as a marker for TDP1 status in certain contexts, which could have implications for cancer treatment approaches.

How should I design immunofluorescence experiments using EFCAB11 antibodies?

For optimal immunofluorescence experiments with EFCAB11 antibodies:

• Sample preparation:

  • Fix cells in 4% formaldehyde/paraformaldehyde in PBS at room temperature for 10-15 minutes

  • Permeabilize with 0.2% Triton X-100 in PBS for approximately 6 minutes

• Antibody concentration and incubation:

  • Use recommended dilutions (typically 0.25-2 μg/mL for immunofluorescence)

  • Incubate primary antibody in PBS containing 5% BSA (PBSB) for 1 hour at room temperature

  • After washing, apply appropriate species-specific fluorophore-conjugated secondary antibody and incubate for 1 hour

• Imaging:

  • Use confocal microscopy with a 63× oil immersion objective for optimal resolution

  • Consider counterstaining with DAPI for nuclear visualization

  • Acquire multiple z-stacks to ensure complete cellular visualization

• Controls:

  • Include negative controls (secondary antibody only)

  • Consider co-staining with organelle markers (e.g., TOM20 for mitochondria, Calnexin for ER) to determine subcellular localization

When analyzing results, focus on subcellular distribution patterns, as EFCAB11 is predicted to have calcium binding functions that may relate to specific cellular compartments.

What are the best practices for Western blot detection of EFCAB11?

For reliable Western blot detection of EFCAB11:

• Sample preparation:

  • Prepare lysates in standard RIPA or similar buffer with protease inhibitors

  • Ensure complete denaturation of protein samples (95°C for 5 minutes in reducing buffer)

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal resolution

  • Perform wet transfer to PVDF membranes for best protein retention

• Antibody application:

  • Block membranes with 5% non-fat milk or BSA in TBST

  • For primary antibody, use rabbit polyclonal anti-EFCAB11 (optimal dilution should be determined experimentally, typically 1:500-1:2000)

  • For loading control, mouse monoclonal anti-β-actin antibody works well

• Detection and troubleshooting:

  • Use either chemiluminescence or fluorescence-based detection systems

  • Expected molecular weight of EFCAB11 is approximately 19-21 kDa

  • If non-specific bands appear, increase blocking time and washing steps

• Validation approaches:

  • Include positive controls (cell lines known to express EFCAB11)

  • Consider using EFCAB11 overexpression constructs as positive controls

  • For negative controls, cell lines with EFCAB11 knockdown or those known to have hypermethylated promoter regions (like HOP_62)

How can I validate the specificity of an EFCAB11 antibody?

To validate EFCAB11 antibody specificity:

• Multiple technique validation:

  • Compare results across Western blotting, immunofluorescence, and ELISA

  • Consistent patterns across techniques suggest specific binding

• Genetic approaches:

  • Use CRISPR/Cas9 to knock out EFCAB11 and confirm loss of signal

  • Alternatively, siRNA knockdown should result in reduced signal intensity

  • Complementation with EFCAB11 expression constructs should restore signal

• Recombinant protein testing:

  • Test antibody against recombinant EFCAB11 protein

  • Some antibodies are validated against protein arrays containing the target protein plus hundreds of non-specific proteins

• Immunoprecipitation:

  • Perform IP followed by mass spectrometry to confirm pulled-down protein identity

  • Reverse IP can further validate the interaction

• Epitope blocking:

  • Pre-incubate antibody with immunizing peptide before application

  • This should abolish specific signal if the antibody is truly specific

High-quality commercial antibodies often include validation data that should be reviewed before purchase .

How should I interpret conflicting immunostaining patterns from different EFCAB11 antibodies?

When facing conflicting immunostaining patterns:

• Analyze epitope differences:

  • Compare the immunogen sequences of each antibody

  • Antibodies targeting different regions may reveal distinct localization patterns if:

    • The protein undergoes post-translational modifications

    • Alternative splicing creates different isoforms

    • The protein has different conformational states

• Evaluate technical parameters:

  • Fixation methods significantly impact epitope accessibility (formaldehyde vs. methanol)

  • Permeabilization approaches affect subcellular compartment access

  • Antibody concentrations influence signal-to-noise ratios

• Consider biological variables:

  • Cell type-specific expression patterns

  • Cell cycle-dependent localization

  • Activation state-dependent changes

• Validation approaches:

  • Use fluorescent protein-tagged EFCAB11 constructs to confirm localization

  • Perform subcellular fractionation followed by Western blotting

  • Apply super-resolution microscopy for detailed localization analysis

• Literature reconciliation:

  • Compare your findings with published data

  • Consider that EFCAB11 may show context-dependent localization related to its calcium-binding function

When presenting conflicting data, clearly document all experimental conditions and discuss potential biological explanations rather than simply attributing differences to antibody quality.

What are common pitfalls in interpreting EFCAB11 expression data in cancer studies?

When analyzing EFCAB11 expression in cancer contexts:

• Bidirectional promoter effects:

  • Remember that EFCAB11 shares a bidirectional promoter with TDP1

  • Changes in EFCAB11 expression may reflect alterations in the shared promoter rather than cancer-specific regulation of EFCAB11 itself

  • Always evaluate TDP1 expression alongside EFCAB11 for comprehensive interpretation

• Epigenetic regulation:

  • Hypermethylation of the shared promoter can silence both genes

  • This epigenetic mechanism has been observed in certain cancer cell lines (e.g., HOP_62)

  • Use methylation-specific PCR or bisulfite sequencing to assess promoter methylation status

• Tissue-specific expression patterns:

  • Baseline EFCAB11 expression varies across tissues

  • Compare tumor samples to matched normal tissue rather than reference databases

  • Consider single-cell analyses to account for heterogeneity within tumor samples

• Technical considerations:

  • RNA-based expression data may not correlate with protein levels

  • Use multiple antibodies targeting different epitopes

  • Confirm findings with orthogonal techniques (qPCR, Western blot, immunohistochemistry)

• Functional relevance:

  • Expression changes alone don't establish causality in cancer progression

  • Correlate findings with clinical outcomes or experimental models

  • Consider that EFCAB11 may be a passenger rather than driver of observed phenotypes

How can I address cross-reactivity issues with EFCAB11 antibodies?

To manage antibody cross-reactivity problems:

• Preventative measures:

  • Select affinity-purified antibodies specifically tested for cross-reactivity

  • Review validation data showing specificity tests against protein arrays (some antibodies are tested against 384 non-specific proteins)

  • Consider monoclonal antibodies for highest specificity, though they may miss certain epitopes

• Experimental approaches to detect cross-reactivity:

  • Perform Western blots under reducing and non-reducing conditions

  • Look for unexpected bands that may indicate cross-reactivity

  • Compare staining patterns in known EFCAB11-negative samples

• Troubleshooting techniques:

  • Increase stringency of washing steps (higher salt concentration, more detergent)

  • Optimize blocking conditions (try different blocking agents like BSA, casein, or commercial blockers)

  • Titrate antibody concentration to find optimal signal-to-noise ratio

• Alternative strategies:

  • Epitope blocking experiments (pre-incubate antibody with immunizing peptide)

  • Use multiple antibodies targeting different regions of EFCAB11

  • CRISPR knockout validation to confirm specificity

• Advanced validation:

  • Mass spectrometry identification of immunoprecipitated proteins

  • Parallel reaction monitoring (PRM) to quantify EFCAB11 peptides

  • Custom antibody development if commercial options prove insufficient

How can I design experiments to investigate EFCAB11's calcium-binding properties?

To study EFCAB11's calcium-binding function:

• Protein expression and purification:

  • Express recombinant EFCAB11 with appropriate tags (His, GST)

  • Design constructs containing full-length protein or isolated EF-hand domains

  • Purify using affinity chromatography followed by size exclusion

• Direct calcium binding assays:

  • Isothermal titration calorimetry (ITC) to measure binding affinity and thermodynamics

  • Microscale thermophoresis (MST) for high-sensitivity binding measurements

  • Calcium overlay assays (45Ca2+ binding to membrane-immobilized protein)

• Structural analysis:

  • Circular dichroism to detect calcium-induced conformational changes

  • Nuclear magnetic resonance (NMR) to identify calcium coordination sites

  • X-ray crystallography of EFCAB11 with and without calcium

• Cellular assays:

  • FRET-based calcium sensors fused to EFCAB11

  • Calcium imaging in cells overexpressing or depleted of EFCAB11

  • Co-localization with calcium channels or calcium-regulated processes

• Mutational analysis:

  • Generate point mutations in predicted EF-hand motifs

  • Assess calcium binding of mutants compared to wild-type protein

  • Evaluate functional consequences of mutations in cellular assays

This multi-faceted approach will clarify whether EFCAB11's predicted calcium binding activity is functional and identify specific calcium-binding properties.

What approaches can I use to study the relationship between EFCAB11 and the microtubule cytoskeleton?

To investigate EFCAB11's role in microtubule organization :

• Co-localization studies:

  • Perform dual immunofluorescence for EFCAB11 and tubulin

  • Use super-resolution microscopy (STED, STORM) for detailed spatial analysis

  • Live-cell imaging with fluorescently tagged EFCAB11 and tubulin

• Biochemical interaction assays:

  • Co-immunoprecipitation of EFCAB11 with tubulin or microtubule-associated proteins

  • In vitro binding assays with purified components

  • Proximity ligation assays to detect interactions in situ

• Functional assays:

  • EFCAB11 knockdown or knockout followed by analysis of:

    • Microtubule stability (nocodazole resistance)

    • Microtubule dynamics (EB1 tracking)

    • Microtubule organization (centrosome function, spindle formation)

  • Rescue experiments with wild-type or mutant EFCAB11

• Cell cycle analysis:

  • Synchronize cells and examine EFCAB11 localization throughout the cell cycle

  • Assess impact of EFCAB11 depletion on mitotic progression

  • Evaluate role in specialized microtubule structures (primary cilia, neuronal processes)

• Calcium dependency:

  • Determine if calcium affects EFCAB11-microtubule interactions

  • Use calcium chelators and ionophores to manipulate cellular calcium levels

  • Compare wild-type EFCAB11 with calcium-binding deficient mutants

These approaches will help establish whether EFCAB11 directly or indirectly influences microtubule organization and dynamics, as suggested by Gene Ontology annotations .

How can I investigate the epigenetic regulation of the EFCAB11-TDP1 bidirectional promoter?

To study the shared promoter of EFCAB11 and TDP1 :

• Promoter methylation analysis:

  • Bisulfite sequencing to determine CpG methylation patterns

  • Methylation-specific PCR for targeted analysis

  • Genome-wide methylation arrays to place findings in broader context

• Chromatin landscape characterization:

  • ChIP-seq for histone modifications (H3K4me3, H3K27ac, H3K9me3)

  • ATAC-seq to assess chromatin accessibility

  • CUT&RUN for high-resolution protein-DNA interaction mapping

• Functional promoter studies:

  • Reporter assays with bidirectional promoter constructs

  • CRISPR-mediated epigenetic editing (dCas9-DNMT for methylation, dCas9-TET1 for demethylation)

  • Deletion or mutation of specific regulatory elements

• Transcription factor binding:

  • ChIP-seq for transcription factors regulating bidirectional promoters

  • Electrophoretic mobility shift assays (EMSA) with nuclear extracts

  • Transcription factor knockdown followed by expression analysis

• Clinical correlation:

  • Analyze promoter methylation in cancer tissues compared to matched normal

  • Correlate EFCAB11/TDP1 expression with promoter methylation status

  • Assess impact of demethylating agents (5-azacytidine) on expression

This comprehensive approach will provide insights into the regulatory mechanisms controlling this bidirectional promoter and potentially identify therapeutic targets for cancers where TDP1 silencing contributes to disease processes .

What strategies can I employ to develop and validate EFCAB11 antibodies with improved specificity for research applications?

For developing highly specific EFCAB11 antibodies:

• Target selection strategies:

  • Perform bioinformatic analysis to identify unique regions with low homology to other proteins

  • Consider regions that are:

    • Surface-exposed in the native protein

    • Evolutionarily conserved (for cross-species reactivity)

    • Outside EF-hand domains (to avoid cross-reactivity with other calcium-binding proteins)

• Antibody generation approaches:

  • Recombinant monoclonal antibody development:

    • Phage display selection against specific epitopes

    • Single B-cell isolation and antibody cloning

  • Traditional hybridoma development with stringent screening

  • Synthetic antibody alternatives (nanobodies, affibodies)

• Validation methodology:

  • Multi-platform testing (immunoblotting, immunohistochemistry, immunoprecipitation)

  • Testing against EFCAB11 knockout cells/tissues as negative controls

  • Cross-reactivity assessment against protein arrays containing homologous proteins

• Epitope engineering:

  • Design chimeric immunogens with carrier proteins

  • Cyclized peptides to mimic conformational epitopes

  • Multi-epitope cocktails to increase specificity

• Advanced antibody engineering:

  • Ultra-stable cytoplasmic antibodies (STANDs) for targeting intracellular EFCAB11

  • Fusion of peptide tags with negative charge for improved stability

  • Site-directed mutagenesis to enhance binding affinity and specificity

This strategic approach combines bioinformatics, protein engineering, and rigorous validation to develop antibodies with exceptional specificity for EFCAB11 research applications.

How can EFCAB11 antibodies be used to investigate potential roles in hepatocellular carcinoma?

Based on EFCAB11's association with hepatocellular carcinoma (HCC) risk factors :

• Expression profiling:

  • Perform immunohistochemistry on tissue microarrays containing HCC samples of various grades and etiologies

  • Compare EFCAB11 expression levels between tumor and adjacent normal tissue

  • Correlate expression with clinicopathological features and patient outcomes

• Mechanistic investigations:

  • Analyze EFCAB11 expression in relation to immune infiltrates in the tumor microenvironment

  • Examine co-expression with inflammatory markers and cytokines

  • Assess relationship with TDP1 expression given their shared bidirectional promoter

• Functional studies:

  • Generate EFCAB11 knockdown and overexpression in HCC cell lines

  • Evaluate effects on proliferation, migration, invasion, and apoptosis

  • Perform xenograft studies to assess impact on tumor growth in vivo

• Biomarker potential:

  • Develop sandwich ELISA using EFCAB11 antibodies for serum/plasma detection

  • Evaluate prognostic value in prospective patient cohorts

  • Assess utility as a companion diagnostic for specific treatments

• Therapeutic targeting:

  • Screen for compounds that modulate EFCAB11 expression or function

  • Investigate epigenetic therapies targeting the EFCAB11-TDP1 promoter

  • Explore potential for antibody-based therapeutic approaches

These approaches could clarify whether EFCAB11 plays a functional role in HCC pathogenesis or serves primarily as a biomarker associated with relevant immune pathways.

What techniques can be employed to study the relationship between EFCAB11 and monoamine metabolism in the central nervous system?

To investigate EFCAB11's association with monoamine metabolite levels :

• Expression mapping in the CNS:

  • Immunohistochemistry/immunofluorescence on brain and spinal cord sections

  • Single-cell RNA sequencing to identify specific neuronal populations expressing EFCAB11

  • Comparison with monoaminergic neuron markers (TH, TPH, DDC)

• CSF analysis approaches:

  • Develop assays to quantify EFCAB11 in cerebrospinal fluid

  • Correlate EFCAB11 levels with monoamine metabolites (5-HIAA, HVA, MHPG)

  • Examine variation across neurological and psychiatric conditions

• Functional studies in neuronal models:

  • EFCAB11 knockdown/overexpression in neuronal cell lines and primary cultures

  • Measure impact on monoamine synthesis, release, and metabolism

  • Calcium imaging to assess relationship between calcium signaling and monoamine regulation

• Animal model investigations:

  • Generate EFCAB11 conditional knockout mice targeting monoaminergic neurons

  • Perform microdialysis to measure neurotransmitter release

  • Behavioral phenotyping focusing on domains regulated by monoamine systems

• Genetic association studies:

  • Analyze SNPs in EFCAB11 in relation to monoamine-related disorders

  • Perform quantitative trait locus analysis linking EFCAB11 variants to CSF monoamine levels

  • Conduct Mendelian randomization studies to assess causality

These methodologies would help determine whether EFCAB11 has a direct functional role in monoamine metabolism or if the association reflects broader involvement in neuronal calcium signaling that indirectly affects monoaminergic systems.

How can new antibody engineering approaches be applied to develop EFCAB11-specific intracellular probes?

Leveraging advances in antibody engineering for intracellular EFCAB11 detection:

• STAND technology application:

  • Ultra-stable cytoplasmic antibodies can be engineered by fusing peptide tags with strong negative charge to anti-EFCAB11 antibody fragments

  • This approach overcomes the problem of antibody aggregation in the reducing environment of the cytoplasm

  • These engineered antibodies maintain functionality without requiring disulfide bridge formation

• Nanobody development:

  • Single-domain antibodies derived from camelid heavy-chain antibodies

  • Their small size (~15 kDa) and high stability make them ideal for intracellular applications

  • Can be expressed directly in cells for live imaging of endogenous EFCAB11

• Intrabody optimization:

  • Convert existing EFCAB11 antibodies to single-chain variable fragments (scFvs)

  • Apply directed evolution to enhance stability in the cytoplasmic environment

  • Incorporate framework mutations that reduce aggregation propensity

• Fusion constructs for live-cell applications:

  • Create fluorescent protein fusions for direct visualization

  • Develop split-fluorescent protein complementation assays to detect EFCAB11 interactions

  • Design FRET-based biosensors to monitor calcium binding by EFCAB11 in real-time

• Delivery strategies:

  • Electroporation of purified antibody fragments

  • Cell-penetrating peptide conjugation

  • Lipid nanoparticle encapsulation for enhanced cellular uptake

These approaches would enable unprecedented visualization and manipulation of endogenous EFCAB11 in living cells, advancing understanding of its dynamic functions and interactions.

What bioinformatic approaches can help predict potential interaction partners for EFCAB11?

To predict EFCAB11 interaction networks:

• Structural prediction and docking:

  • Generate 3D structural models using AlphaFold or similar tools

  • Perform molecular docking simulations with potential partners

  • Identify interaction interfaces based on electrostatic and hydrophobic properties

• Co-expression analysis:

  • Mine RNA-seq databases for genes showing correlated expression patterns

  • Perform weighted gene correlation network analysis (WGCNA)

  • Identify tissue-specific co-expression clusters

• Evolutionary approaches:

  • Phylogenetic profiling to identify proteins with similar evolutionary patterns

  • Analysis of correlated mutations suggesting co-evolution of interaction interfaces

  • Comparison with other EF-hand domain-containing proteins

• Text mining and knowledge integration:

  • Natural language processing of scientific literature

  • Integration of pathway databases and protein interaction networks

  • Analysis of functional annotations and cellular compartmentalization

• Machine learning applications:

  • Train models on known calcium-binding protein interactions

  • Integrate multiple data types (structure, expression, localization)

  • Predict probability of protein-protein interactions based on learned features

These computational approaches can generate testable hypotheses about EFCAB11's functional partners, guiding subsequent experimental validation using co-immunoprecipitation, proximity labeling, or other interaction detection methods.

How might EFCAB11 antibodies be adapted for therapeutic applications based on recent advances in antibody therapeutics?

Exploring therapeutic potential of EFCAB11 antibodies:

• Intracellular antibody delivery strategies:

  • Applying STAND technology to create stable cytoplasmic antibodies

  • Lipid nanoparticle encapsulation for cellular delivery

  • Cell-penetrating peptide conjugation for enhanced uptake

• Targeted protein degradation approaches:

  • Development of EFCAB11-targeting PROTACs (proteolysis targeting chimeras)

  • Antibody-PROTAC conjugates to induce selective EFCAB11 degradation

  • E3 ubiquitin ligase recruitment to EFCAB11 via engineered antibodies

• Immune-directed therapies:

  • Antibody-drug conjugates if EFCAB11 shows cell-surface expression in disease states

  • CAR-T cell approaches for cancers with aberrant EFCAB11 expression

  • Bispecific antibodies linking EFCAB11-expressing cells to immune effectors

• Modulation of calcium signaling:

  • Antibodies designed to block or enhance EFCAB11 calcium binding

  • Targeting EFCAB11-mediated calcium signaling in specific disease contexts

  • Allosteric modulators of EFCAB11 function

• Epigenetic targeting:

  • Approaches aimed at reversing silencing of the EFCAB11-TDP1 bidirectional promoter

  • Antibody-guided epigenetic modifiers to specific genomic loci

  • Combined targeting with TDP1-directed therapies in cancer contexts

While current evidence for EFCAB11 as a therapeutic target remains preliminary, these approaches illustrate how advances in antibody engineering technology could potentially be applied if EFCAB11 emerges as a clinically relevant target.