CEACAM16 Antibody, Biotin conjugated

What is CEACAM16 and why is it significant for auditory research?

CEACAM16 (Carcinoembryonic antigen-related cell adhesion molecule 16) is a highly conserved protein specifically expressed in the inner ear that plays a crucial role in mammalian hearing. Its significance stems from its deposition in the tectorial membrane of the cochlea between postnatal days 12 and 15, coinciding with the onset of hearing in mice. CEACAM16 deficiency leads to progressive hearing impairment characterized by raised thresholds for frequencies below 10 kHz and above 22 kHz. This hearing impairment pattern has also been observed in humans with DFNA4 non-syndromic autosomal dominant hearing loss carrying CEACAM16 mutations, highlighting its critical role in maintaining normal auditory function . Research targeting CEACAM16 is therefore essential for understanding both normal hearing mechanisms and progressive hearing loss disorders.

What are the technical specifications of CEACAM16 Antibody, Biotin conjugated?

The CEACAM16 Antibody, Biotin conjugated (e.g., SKU: QA16985) is a polyclonal antibody raised in rabbits against recombinant Human Carcinoembryonic antigen-related cell adhesion molecule 16 protein (specifically amino acids 323-414). The antibody has been purified using Protein G chromatography (>95% purity) and subsequently conjugated with biotin. It is formulated as a liquid in a buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4. It specifically reacts with human CEACAM16 and has been validated for ELISA applications . The biotin conjugation enables versatile detection systems utilizing streptavidin-based secondary reagents.

How should CEACAM16 Antibody, Biotin conjugated be stored and handled to maintain optimal activity?

For optimal preservation of activity, CEACAM16 Antibody, Biotin conjugated should be stored at -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can lead to antibody degradation and loss of binding affinity . When working with the antibody, aliquoting into single-use volumes is recommended to prevent multiple freeze-thaw events. During experiments, the antibody should be kept on ice when not in use, and exposure to direct light should be minimized to prevent photobleaching of the biotin conjugate. Additionally, maintaining sterility is important to prevent microbial contamination that could degrade the antibody or interfere with experimental results.

How can CEACAM16 Antibody, Biotin conjugated be used in ELISA applications?

For ELISA applications using CEACAM16 Antibody, Biotin conjugated, researchers should follow these methodological steps:

• Coat ELISA plates with the target antigen (recombinant CEACAM16 or tissue lysates containing CEACAM16) in carbonate buffer (pH 9.6) overnight at 4°C

• Block non-specific binding sites with 5% non-fat milk or BSA in PBS for 1-2 hours at room temperature

• Incubate with the biotin-conjugated CEACAM16 antibody (typically at 1:1000 dilution) for 1-2 hours at room temperature

• Detect using streptavidin-HRP (1:5000 to 1:10000) for 1 hour at 37°C

• Develop with TMB substrate and measure absorbance at 450 nm

This methodology has been validated for detecting human CEACAM16 with high specificity . For quantification, a standard curve should be generated using purified recombinant CEACAM16 protein. When analyzing samples with unknown CEACAM16 concentrations, multiple dilutions should be tested to ensure readings fall within the linear range of the standard curve.

What protocols should be followed for Western blot analysis using CEACAM16 antibodies?

For effective Western blot detection of CEACAM16 using biotin-conjugated antibodies, the following optimized protocol is recommended:

• Extract proteins from samples using appropriate lysis buffers containing protease inhibitors

• For secreted CEACAM16, concentrate culture medium using ultrafiltration tubes before analysis

• Separate proteins via SDS-PAGE (typically 10-12%) and transfer to PVDF membranes

• Block membranes with 5% non-fat milk in TBST for 2 hours at room temperature

• Incubate with primary CEACAM16 antibody at 1:1000 dilution overnight at 4°C

• Wash thoroughly with TBST buffer

• Incubate with streptavidin-HRP (for biotin-conjugated antibodies) at appropriate dilution

• Develop using chemiluminescence detection

Under reducing conditions, expect to detect a band of approximately 53 kDa corresponding to CEACAM16 protein . For analysis of potential oligomeric forms, perform parallel runs under non-reducing conditions (omitting reducing agents like β-mercaptoethanol or DTT), which can reveal higher molecular weight complexes formed through unpaired cysteine interactions .

How can CEACAM16 antibodies be used to investigate oligomerization of CEACAM16?

To investigate CEACAM16 oligomerization, which occurs via unpaired cysteines and is critical for its function in the tectorial membrane, implement the following approach:

• Perform Western blot analysis under both reducing and non-reducing conditions:

  • Non-reducing conditions: Omit reducing agents in sample buffer

  • Reducing conditions: Include β-mercaptoethanol or DTT in sample buffer

• Compare molecular weight patterns between the two conditions:

  • Under reducing conditions, expect a predominant 53 kDa band

  • Under non-reducing conditions, look for higher molecular weight bands indicating oligomers

• For further validation, perform crosslinking experiments using:

  • Membrane-impermeable crosslinkers for cell surface proteins

  • Formaldehyde or glutaraldehyde for fixed tissue samples

• Immunoprecipitation can confirm interaction partners by:

  • Using biotin-conjugated CEACAM16 antibodies to pull down protein complexes

  • Identifying binding partners via mass spectrometry

Research has demonstrated that CEACAM16 can form higher-order structures with other tectorial membrane proteins such as α-tectorin and β-tectorin, influencing the physical properties of the tectorial membrane . These oligomerization studies are particularly relevant for understanding how CEACAM16 mutations affect protein structure and function in hearing disorders.

How can CEACAM16 Antibody be used to study the role of CEACAM16 in hearing loss models?

For investigating CEACAM16's role in hearing loss models, researchers can implement these methodological approaches:

• Immunohistochemistry of cochlear sections:

  • Use biotin-conjugated CEACAM16 antibodies followed by streptavidin-fluorophore detection

  • Compare wild-type tissues with CEACAM16 knockout or mutant models

  • Assess tectorial membrane morphology and CEACAM16 deposition patterns

• Correlative studies between CEACAM16 expression and auditory function:

  • Perform auditory brainstem response (ABR) testing at various frequencies

  • Collect cochlear tissues for immunostaining with CEACAM16 antibodies

  • Analyze correlation between hearing thresholds and CEACAM16 distribution/intensity

• Temporal expression studies during cochlear development:

  • Collect cochlear tissues at different developmental stages (particularly around P12-P15)

  • Use CEACAM16 antibodies to track protein deposition in the tectorial membrane

  • Correlate with the onset of hearing function

Research has shown that CEACAM16 is deposited in the tectorial membrane between postnatal days 12 and 15, coinciding with hearing onset in mice. In CEACAM16 knockout mice, tectorial membranes are more frequently stretched out compared to wild-type mice where they are typically contracted and detached from outer hair cells . This methodological approach can help elucidate the mechanism by which CEACAM16 mutations lead to progressive hearing loss.

What approaches can be used to investigate CEACAM16 protein-protein interactions in the inner ear?

To investigate CEACAM16 protein-protein interactions in the inner ear, implement these advanced methodological approaches:

• Co-immunoprecipitation studies:

  • Use biotin-conjugated CEACAM16 antibodies with streptavidin beads to pull down protein complexes

  • Identify interaction partners (such as α-tectorin and β-tectorin) by Western blot or mass spectrometry

  • Compare interaction profiles between wild-type and mutant CEACAM16 variants

• Proximity ligation assays in tissue sections:

  • Utilize CEACAM16 antibodies alongside antibodies against potential binding partners

  • Visualize protein-protein interactions in situ with subcellular resolution

  • Quantify interaction signals across different cochlear regions or developmental stages

• In vitro binding assays:

  • Express recombinant CEACAM16 domains (particularly the N1 and N2 immunoglobulin-like domains)

  • Test homotypic and heterotypic interactions using surface plasmon resonance or ELISA

  • Evaluate effects of disease-causing mutations on binding affinities

Research has demonstrated that CEACAM16 can engage in trans interactions through its carboxyl-terminal immunoglobulin variable-like N2 domain, and can form oligomers via unpaired cysteines . These protein-protein interactions likely facilitate the formation of higher-order structures with other tectorial membrane proteins, influencing cochlear mechanics and auditory function.

How can mini-gene splicing assays be used to evaluate the functional impact of CEACAM16 splice-site variants?

For evaluating the functional consequences of CEACAM16 splice-site variants, mini-gene splicing assays provide valuable insights through these methodological steps:

• Construction of mini-gene vectors:

  • Amplify wild-type CEACAM16 exons and flanking intronic sequences by PCR

  • Ligate amplicons into expression vectors like pET01 Exontrap

  • Introduce the splice-site variants using site-directed mutagenesis

• Cell-based expression analysis:

  • Transfect wild-type and mutant mini-genes into appropriate cell lines (e.g., COS7, HEK293)

  • Extract total RNA 36-48 hours post-transfection

  • Synthesize cDNA using vector-specific primers

  • Amplify splicing products by PCR and visualize on agarose gels

• Sequencing and analysis:

  • Extract and sequence PCR products to identify splicing alterations

  • Analyze potential effects on reading frame and protein structure

  • Correlate splicing defects with clinical phenotypes

This approach has revealed that certain CEACAM16 variants (like c.37G>T) can cause exon skipping resulting in loss of the start codon, while others (like c.662-1G>C) activate cryptic splice sites leading to frameshift mutations . These findings provide mechanistic explanations for how CEACAM16 mutations contribute to post-lingual progressive hearing impairment through loss-of-function mechanisms.

How does research on CEACAM16 antibodies contribute to understanding hereditary hearing loss?

Research utilizing CEACAM16 antibodies has significantly advanced our understanding of hereditary hearing loss through these key contributions:

• Phenotype-genotype correlations:

  • Immunohistochemical studies using CEACAM16 antibodies have revealed abnormal tectorial membrane morphology in individuals with CEACAM16 mutations

  • These structural changes correlate with specific audiometric patterns showing elevated thresholds for frequencies below 10 kHz and above 22 kHz

• Mutation impact assessment:

  • Western blot analysis of mutant CEACAM16 proteins has demonstrated altered expression levels and oligomerization patterns

  • ELISA quantification using biotin-conjugated antibodies has shown significantly higher levels of certain mutant CEACAM16 proteins compared to wild-type, indicating potential pathogenic mechanisms

• Development of diagnostic approaches:

  • CEACAM16 antibodies enable detection of protein expression and localization in patient-derived samples

  • This facilitates correlation of protein levels with genetic variants and clinical presentation

Studies have identified that both heterozygous missense mutations (associated with DFNA4) and homozygous splice-altering variants in CEACAM16 result in post-lingual progressive hearing impairment . The use of antibodies in these investigations has been crucial for establishing CEACAM16's role in auditory function and understanding the molecular mechanisms underlying CEACAM16-associated hearing disorders.

What methodological approaches can address data discrepancies in CEACAM16 antibody studies?

When facing data discrepancies in CEACAM16 antibody studies, implement these methodological solutions:

• Antibody validation strategies:

  • Perform parallel experiments with multiple CEACAM16 antibodies targeting different epitopes

  • Include CEACAM16 knockout tissues or cells as negative controls

  • Verify specificity through pre-adsorption tests with recombinant CEACAM16

• Expression system considerations:

  • Compare results across different expression systems (bacterial, mammalian, insect cells)

  • Account for potential post-translational modifications in mammalian versus non-mammalian systems

  • Consider the impact of tags or fusion partners on protein folding and antibody recognition

• Protocol standardization:

  • Systematically optimize antibody concentrations, incubation times, and buffer conditions

  • Perform side-by-side comparisons of different detection methods (direct vs. indirect)

  • Document detailed protocols to improve reproducibility

One study noted limitations in detecting CEACAM16 oligomers using commercial antibody kits , while another successfully demonstrated oligomerization via unpaired cysteines . Such discrepancies might stem from differences in experimental conditions, sample preparation methods, or the specific epitopes recognized by different antibodies. Implementing rigorous validation and standardization procedures is essential for resolving these inconsistencies and generating reliable data.

How can CEACAM16 antibodies be applied in translational research models of hearing loss?

For translational research applications in hearing loss models, CEACAM16 antibodies can be utilized through these methodological approaches:

• Therapeutic antibody development:

  • Engineer modified antibodies that could stabilize CEACAM16 structure in cases of destabilizing mutations

  • Develop antibodies that could block aberrant interactions of mutant CEACAM16

  • Test these therapeutic candidates in animal models before clinical translation

• Biomarker development:

  • Establish ELISA-based assays using biotin-conjugated CEACAM16 antibodies to detect CEACAM16 levels in accessible fluids

  • Correlate CEACAM16 levels with hearing function measures and disease progression

  • Evaluate potential as diagnostic or prognostic indicators for hearing disorders

• In vivo imaging:

  • Adapt biotin-conjugated antibodies for in vivo imaging in animal models

  • Track CEACAM16 distribution and dynamics during development and in disease states

  • Monitor therapeutic responses in real-time

Research has established that CEACAM16's role in maintaining tectorial membrane integrity is critical for proper hearing function across an extended frequency range . Translational applications of CEACAM16 antibodies could therefore target the restoration of normal tectorial membrane properties in cases of CEACAM16 dysfunction, potentially leading to novel therapeutic strategies for certain forms of progressive hearing loss.

What are the relative advantages of using biotin-conjugated versus unconjugated CEACAM16 antibodies?

The choice between biotin-conjugated and unconjugated CEACAM16 antibodies involves several methodological considerations:

How do different antibody-based methods compare for detecting CEACAM16 expression and localization?

The following methodological comparison highlights the strengths and limitations of different antibody-based approaches for CEACAM16 detection:

For cochlear studies, immunohistochemistry has been particularly valuable in demonstrating CEACAM16 localization in interdental and Deiters cells and its deposition in the tectorial membrane . Western blot analysis under reducing and non-reducing conditions has effectively revealed CEACAM16 oligomerization properties. ELISA has proven useful for quantitative analysis of wild-type versus mutant CEACAM16 protein levels . The choice of method should be guided by the specific research question, with consideration of required sensitivity and spatial information.

What emerging technologies might enhance CEACAM16 antibody applications in hearing research?

Several emerging technologies offer promising enhancements for CEACAM16 antibody applications in hearing research:

• Single-cell analysis methods:

  • Single-cell Western blotting to analyze CEACAM16 expression in rare cell populations

  • Mass cytometry (CyTOF) with CEACAM16 antibodies for multiparameter analysis of cochlear cells

  • Spatial transcriptomics combined with CEACAM16 immunostaining to correlate protein localization with gene expression profiles

• Advanced imaging technologies:

  • Super-resolution microscopy (STORM, PALM) to visualize CEACAM16 nanoscale organization in the tectorial membrane

  • Expansion microscopy to achieve improved spatial resolution of CEACAM16 distribution

  • Intravital imaging with minimally invasive techniques to study CEACAM16 dynamics in live animal models

• Antibody engineering approaches:

  • Development of single-domain antibodies (nanobodies) against CEACAM16 for improved tissue penetration

  • Bispecific antibodies targeting CEACAM16 and interacting partners simultaneously

  • Conformation-specific antibodies to distinguish between different oligomeric states

These technologies would enable more precise characterization of CEACAM16's role in tectorial membrane structure and function, potentially revealing new mechanisms by which CEACAM16 mutations lead to progressive hearing loss. The application of these advanced methods could significantly enhance our understanding of CEACAM16's involvement in both normal hearing and pathological states.

How might antibody-based techniques contribute to developing therapeutics for CEACAM16-related hearing disorders?

Antibody-based techniques have significant potential for therapeutic development in CEACAM16-related hearing disorders through these methodological approaches:

• Stabilization of mutant CEACAM16 protein:

  • Development of conformation-specific antibodies that bind and stabilize mutant CEACAM16

  • Engineering of intrabodies (intracellular antibodies) to aid proper folding of mutant proteins

  • Creation of antibody-based chaperones to prevent degradation of partially functional mutants

• Gene therapy enhancement:

  • Use antibodies to track gene therapy delivery and expression in cochlear structures

  • Develop antibody-guided nanoparticles for targeted delivery of CEACAM16 gene constructs

  • Monitor therapeutic efficacy through antibody-based quantification of CEACAM16 restoration

• Early detection and intervention:

  • Create diagnostic panels using CEACAM16 antibodies to detect subclinical protein abnormalities

  • Develop screening assays for at-risk populations carrying CEACAM16 variants

  • Monitor disease progression and therapeutic response through quantitative antibody-based assays

Research has established that loss-of-function mutations in CEACAM16 result in post-lingual progressive hearing impairment . As such, therapeutic approaches may focus on maintaining or restoring sufficient levels of functional CEACAM16 in the tectorial membrane. Antibody-based techniques offer powerful tools for both therapeutic development and monitoring treatment efficacy in these specialized hearing disorders.