Ctl2 Antibody

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Product Specs

Buffer
**Preservative:** 0.03% Proclin 300
**Constituents:** 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Form
Liquid
Synonyms
CTL-like protein 2 CG11880
Target Names
Ctl2 Antibody
Uniprot No.

Target Background

Database Links

KEGG: dme:Dmel_CG11880

STRING: 7227.FBpp0084740

UniGene: Dm.10965

Protein Families
CTL (choline transporter-like) family
Subcellular Location
Membrane; Multi-pass membrane protein.

Q&A

What is CTL2 and why is it significant in autoimmune hearing loss research?

CTL2 (Choline transporter-like protein 2) is a 68-72 kDa inner ear membrane glycoprotein that has been identified as a candidate target antigen in autoimmune hearing loss (AIHL). This protein is a member of the solute carrier family with the designation SLC44A2 and contains 10-11 transmembrane domains, one of which is implicated in lipid transport and metabolism. The significance of CTL2 in AIHL research stems from observations that antibodies binding to this protein can cause hearing loss, and patients with AIHL have demonstrated antibodies that bind to inner ear supporting cells with the same distribution pattern as anti-CTL2 antibodies .

The human version of CTL2 has a canonical amino acid length of 706 residues and a protein mass of approximately 80.1 kilodaltons, although three isoforms have been identified. It functions in NF-kappaB signaling pathways and transmembrane transport and is notably expressed in many tissues, such as the duodenum and endometrium .

How does recombinant human CTL2 protein present on Western blots?

When analyzing recombinant human CTL2 (rHuCTL2) protein on Western blots, researchers should expect to observe a characteristic pattern of three distinct bands:

  • A core protein band at approximately 62 kDa

  • Two N-glycosylated bands at approximately 66 kDa and 70 kDa

This migration pattern differs slightly from native guinea pig CTL2, which typically migrates as two protein bands of 68 and 72 kDa. The differences in migration patterns between recombinant human CTL2 and native guinea pig CTL2 highlight the importance of species-specific considerations when interpreting Western blot results .

What validation techniques should be employed before using CTL2 antibodies in experiments?

Validating CTL2 antibodies is crucial for ensuring experimental rigor and reproducibility. Researchers should implement the following validation approaches:

  • Western blotting verification: Confirm antibody specificity by detecting bands at the expected molecular weights (62-70 kDa for recombinant human CTL2) .

  • Positive and negative controls: Include appropriate tissue or cell lysates known to express or not express CTL2.

  • Cross-validation with multiple antibodies: When possible, verify findings using different antibodies targeting distinct epitopes of CTL2. For example, using antibodies targeting the N-terminal region (anti CTL2-NT), the third outer loop (CTL2-TOL), and the C-terminus (anti CTL2-CT) .

  • Knockout/knockdown controls: When available, use samples from knockout models or cells with knockdown of CTL2 to confirm specificity.

  • Immunoprecipitation validation: Perform immunoprecipitation followed by mass spectrometry to confirm the identity of the pulled-down protein.

These validation steps are crucial as it is estimated that 35% of unreproducible studies may be due to biological reagents, including antibodies .

What optimization strategies are recommended for immunohistochemistry using CTL2 antibodies?

For optimal immunohistochemistry (IHC) results with CTL2 antibodies, researchers should consider the following methodological approaches:

  • Fixation optimization: Test different fixation protocols (e.g., paraformaldehyde, methanol, acetone) to determine which best preserves CTL2 epitopes while maintaining tissue morphology.

  • Antigen retrieval assessment: Compare different antigen retrieval methods (heat-induced vs. enzymatic) to enhance antibody accessibility to CTL2 epitopes.

  • Blocking optimization: Evaluate different blocking solutions to reduce background staining, which is particularly important for membrane proteins like CTL2.

  • Antibody dilution series: Perform a titration series to determine the optimal antibody concentration that maximizes specific signal while minimizing background.

  • Secondary antibody selection: Choose secondary antibodies with minimal cross-reactivity to the species being studied.

  • Inclusion of appropriate controls:

    • Positive tissue controls (known to express CTL2)

    • Negative controls (tissues known not to express CTL2)

    • Technical controls (omitting primary antibody)

How can CTL2 antibodies be utilized to study autoimmune hearing loss mechanisms?

CTL2 antibodies serve as valuable tools for investigating autoimmune hearing loss (AIHL) mechanisms through several advanced approaches:

  • Patient autoantibody detection: rHuCTL2 can be used as a substrate for testing patient sera to detect autoantibodies. Studies have shown that approximately 50% of AIHL patients with antibody to the 68-72 kDa inner-ear protein or to supporting cells also have antibody to rHuCTL2 .

  • Correlation with treatment response: Research has indicated that patients with antibody to rHuCTL2 may respond better to corticosteroid treatment. In one study, four out of four patients with antibody to rHuCTL2 responded to corticosteroids, whereas 50% of patients lacking antibody to rHuCTL2 did not respond .

  • In vitro modeling: CTL2 antibodies can be used to develop in vitro models of autoimmune damage to inner ear cells, allowing for mechanistic studies and potential therapeutic screening.

  • Immunofluorescence mapping: Using CTL2 antibodies for immunofluorescence studies can help map the distribution of CTL2 in the inner ear and identify specific cell types that might be targeted in AIHL.

  • Cross-species comparative studies: Comparing CTL2 antibody binding patterns across species can provide evolutionary insights into conservation of this protein and its functions.

What are the considerations for distinguishing between different isoforms of CTL2 using antibodies?

When investigating different isoforms of CTL2, researchers should consider the following methodological approaches:

  • Epitope-specific antibodies: Select antibodies that target regions unique to specific isoforms. Research has identified three isoforms of human CTL2 , and antibodies raised against distinct regions can help differentiate between them.

  • Western blot analysis: Use high-resolution SDS-PAGE to separate the different isoforms based on their molecular weights. The rHuCTL2 protein migrates as three bands (62, 66, and 70 kDa) , which may represent different isoforms or post-translational modifications.

  • Two-dimensional gel electrophoresis: This technique can help separate isoforms based on both molecular weight and isoelectric point.

  • Immunoprecipitation followed by mass spectrometry: This approach can identify specific isoforms and post-translational modifications with high accuracy.

  • Isoform-specific primers for RT-PCR validation: Complement antibody-based detection with mRNA analysis using isoform-specific primers.

  • Glycosylation analysis: Since CTL2 is known to be N-glycosylated, treatment with deglycosylation enzymes followed by Western blotting can help distinguish between glycosylated and non-glycosylated forms .

Isoform Detection MethodAdvantagesLimitations
Epitope-specific antibodiesDirect detection of proteinMay cross-react with similar epitopes
Western blot analysisVisual separation of isoformsLimited resolution for similar-sized isoforms
Two-dimensional gel electrophoresisSeparates based on two propertiesTechnical complexity and expertise required
Mass spectrometryHigh accuracy identificationExpensive equipment and expertise required
RT-PCR validationConfirms expression at mRNA levelDoesn't confirm protein abundance or modifications
Glycosylation analysisIdentifies post-translational modificationsMay not distinguish between all isoforms

How should researchers address non-specific binding of CTL2 antibodies?

Non-specific binding is a common challenge when working with antibodies, including those targeting CTL2. To address this issue, researchers should consider implementing the following methodological approaches:

  • Optimize blocking conditions: Test different blocking agents (BSA, normal serum, commercial blockers) at various concentrations and incubation times.

  • Adjust antibody concentration: Perform titration experiments to identify the optimal antibody dilution that maximizes specific signal while minimizing background.

  • Increase washing stringency: Use more detergent (e.g., Tween-20, Triton X-100) in wash buffers or increase the number and duration of washing steps.

  • Pre-adsorption controls: Pre-incubate the antibody with purified target protein (if available) to confirm specificity. The signal should be reduced or eliminated if the antibody is specific.

  • Use alternative fixation methods: Different fixation protocols can affect epitope accessibility and non-specific binding. Compare paraformaldehyde, methanol, acetone, or combinations.

  • Employ alternative detection systems: Test different secondary antibodies or detection reagents that may offer improved signal-to-noise ratios.

  • Validate with knockout/knockdown controls: Utilize CTL2 knockout or knockdown samples to confirm antibody specificity .

What approaches can verify CTL2 antibody specificity across different applications?

Verifying antibody specificity is crucial for ensuring reliable experimental results. For CTL2 antibodies, researchers should implement the following verification approaches:

  • Multi-technique cross-validation: Verify findings using multiple techniques (Western blot, IHC, ELISA, flow cytometry) to ensure consistent results across different applications.

  • Multiple antibody validation: Test several antibodies targeting different epitopes of CTL2. For instance, antibodies raised against the N-terminal region (CTL2-NT), the third outer loop (CTL2-TOL), and the C-terminus (CTL2-CT) can provide complementary information .

  • Genetic validation:

    • Use siRNA or shRNA knockdown of CTL2

    • Employ CRISPR/Cas9 knockout models

    • Utilize overexpression systems

  • Antigen competition assays: Pre-incubate the antibody with excess purified CTL2 protein or the specific peptide used for immunization before application.

  • Mass spectrometry validation: Confirm the identity of immunoprecipitated proteins using mass spectrometry.

  • Species cross-reactivity assessment: Test the antibody against CTL2 from different species to establish conservation and specificity .

  • Consult validation databases: Utilize resources such as Antibodypedia or The Antibody Registry to assess previously reported validation data .

How do CTL2 antibody detection methods correlate with clinical outcomes in autoimmune hearing loss?

Research has demonstrated important correlations between CTL2 antibody detection and clinical outcomes in autoimmune hearing loss:

  • Treatment response prediction: Studies have shown that patients with antibodies to rHuCTL2 may have better responses to corticosteroid treatment. In one study, all patients (4/4) with antibody to rHuCTL2 responded to corticosteroids, whereas only 50% (4/8) of patients lacking antibody to rHuCTL2 responded to treatment .

  • Diagnostic potential: Detection of antibodies to rHuCTL2 in patient sera may serve as a biomarker for certain forms of autoimmune hearing loss. Research has demonstrated that 50% of AIHL patients with antibody to the 68-72 kDa inner-ear protein or to supporting cells also have antibody to rHuCTL2 .

  • Disease subtype identification: CTL2 antibody detection may help classify AIHL into subtypes based on autoantibody profiles, potentially leading to more tailored treatment approaches.

  • Monitoring disease activity: Serial measurement of CTL2 antibody levels could potentially be used to monitor disease activity and treatment efficacy over time.

  • Research applications: The ability to detect CTL2 antibodies provides a valuable tool for investigating disease mechanisms, potentially leading to new therapeutic targets.

The development of standardized, reliable CTL2 antibody detection methods could significantly improve the diagnosis and management of autoimmune hearing loss.

What novel approaches are being explored for CTL2 antibody-based diagnostics in hearing disorders?

Several innovative approaches are being investigated for CTL2 antibody-based diagnostics in hearing disorders:

  • Recombinant protein-based assays: Optimization of recombinant human CTL2 (rHuCTL2) production in insect cells using baculovirus expression systems allows for efficient substrate production for antibody testing. The insect cell system permits high-level expression of large membrane proteins like CTL2 in quantities sufficient for immunoassay development .

  • Multiplex autoantibody profiling: Combining CTL2 antibody detection with other inner ear autoantibody markers to create comprehensive autoimmune profiles for hearing disorders.

  • Point-of-care testing development: Exploring rapid diagnostic platforms that could potentially be used in clinical settings for quick screening of CTL2 antibodies.

  • Epitope mapping approaches: Identifying specific epitopes recognized by pathogenic autoantibodies in patients with AIHL could lead to more precise diagnostic tests.

  • Imaging techniques: Development of labeled CTL2 antibodies for potential use in imaging studies to visualize affected tissues.

  • Functional antibody assays: Beyond mere detection, assessing the functional impact of patient-derived CTL2 antibodies on cellular models to correlate antibody activity with disease severity.

How might advanced technologies enhance CTL2 antibody research and applications?

Emerging technologies offer promising opportunities to advance CTL2 antibody research:

  • Single-cell antibody screening: Technologies that allow for screening of antibody binding at the single-cell level can provide insights into cellular heterogeneity in CTL2 expression and autoantibody binding.

  • CRISPR-based validation: CRISPR/Cas9 gene editing can create precise knockout models for definitive antibody validation, enhancing the specificity of CTL2 antibody-based assays.

  • Recombinant antibody engineering: Development of recombinant antibodies with enhanced specificity and affinity for CTL2, including humanized antibodies for potential therapeutic applications.

  • Microfluidic platforms: Integration of CTL2 antibody assays into microfluidic platforms could allow for high-throughput screening of patient samples with minimal reagent consumption.

  • Live-cell imaging techniques: Advanced microscopy methods can monitor CTL2 protein dynamics in real-time, providing insights into its functional roles.

  • Proteomics integration: Combining antibody-based detection with mass spectrometry-based proteomics can provide comprehensive characterization of CTL2 protein interactions and modifications .

  • AI-assisted epitope prediction: Machine learning algorithms can help predict optimal epitopes for generating highly specific CTL2 antibodies.

What are the potential therapeutic applications of CTL2 antibodies beyond diagnostic use?

CTL2 antibodies show promise for therapeutic applications beyond their diagnostic utility:

  • Targeted immunotherapy: Development of neutralizing antibodies against pathogenic CTL2 autoantibodies could potentially treat autoimmune hearing loss.

  • Drug delivery systems: CTL2 antibodies could be used to deliver therapeutic agents specifically to cells expressing this protein.

  • Antibody-drug conjugates: Coupling cytotoxic agents to CTL2 antibodies could potentially target cells overexpressing CTL2 in certain pathological conditions.

  • Regenerative medicine applications: CTL2 antibodies might help identify and isolate specific cell populations for regenerative approaches in inner ear disorders.

  • Monitoring therapy response: Serial measurement of CTL2 autoantibody levels could help assess the efficacy of immunosuppressive therapies in AIHL.

  • Prevention strategies: Understanding the epitopes recognized by pathogenic CTL2 autoantibodies could lead to the development of preventative approaches for high-risk individuals.

These potential therapeutic applications highlight the importance of continued research into CTL2 antibodies and their interactions with the immune system in both health and disease.

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