COL23A1 Antibody

What is COL23A1 and what cellular functions does it serve?

COL23A1 (Collagen Type XXIII Alpha 1) is a transmembrane collagen primarily localized in the cell membrane. The canonical human protein consists of 540 amino acid residues with a calculated molecular weight of approximately 52 kDa, though it typically appears at 68-75 kDa in experimental conditions due to post-translational modifications . COL23A1 is primarily involved in extracellular matrix organization and cell-cell adhesion processes .

Recent research has demonstrated that COL23A1 plays significant roles in:

• Cell adhesion and anchorage-independent growth in cancer cell lines

• Enhanced susceptibility to HSV-1 viral infection in keratinocytes

• Potential mediation of cell-cell and cell-matrix interactions that may promote metastasis

What types of COL23A1 antibodies are most commonly used in research settings?

COL23A1 antibodies are available in several configurations based on:

Notable antibodies include mouse monoclonal antibodies targeting AA 338-410 for human samples (clone 2E10) and rabbit polyclonal antibodies with broader species reactivity .

What experimental applications are validated for COL23A1 antibodies?

The following applications have been experimentally validated:

• Western Blotting (WB): Successfully detected COL23A1 at 68-75 kDa in mouse thymus tissue and human lung carcinoma cell lines

• Immunohistochemistry (IHC): Validated for human colon tissue and adenocarcinoma samples with specific cytoplasmic staining

• Enzyme-Linked Immunosorbent Assay (ELISA): Multiple antibodies are optimized for ELISA applications

• Immunofluorescence (IF): Several polyclonal antibodies show strong reactivity in IF applications

• Immunoprecipitation (IP): Limited antibodies are validated for this application

What are the optimal protocols for COL23A1 detection in Western Blotting experiments?

For optimal COL23A1 detection by Western Blot:

• Sample Preparation:

  • Mouse thymus tissue or human lung carcinoma cell lines (e.g., NCI-H460) serve as positive controls

  • Use RIPA buffer supplemented with protease inhibitors

  • Run under reducing conditions using Immunoblot Buffer Group 1

• SDS-PAGE and Transfer:

  • Use PVDF membrane for optimal protein binding

  • The calculated molecular weight of COL23A1 is 52 kDa, but expect bands at approximately 68-75 kDa due to post-translational modifications

  • In some lung carcinoma samples, both 75 kDa and 60 kDa bands may be observed

• Antibody Incubation:

  • Primary antibody dilution: 1:500-1:1000 (for polyclonal antibodies)

  • For monoclonal antibodies, use approximately 2 μg/mL concentration

  • Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit IgG

• Detection:

  • Enhanced chemiluminescence (ECL) is suitable for visualization

  • Validate specificity with known positive controls

What considerations are important for immunohistochemical detection of COL23A1?

For successful IHC detection of COL23A1:

• Tissue Preparation:

  • Paraffin-embedded tissue sections are suitable

  • Human colon tissue and adenocarcinoma samples serve as positive controls

• Antigen Retrieval:

  • Recommended: Heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0)

  • Alternative: Citrate buffer (pH 6.0) may be used if Tris-EDTA proves suboptimal

• Antibody Incubation:

  • For polyclonal antibodies: Use 1:500-1:2000 dilution

  • For monoclonal antibodies: Use 25 μg/mL concentration

  • For best results with monoclonal antibodies, incubate overnight at 4°C

• Detection System:

  • HRP-DAB system shows excellent results for visualization (brown staining)

  • Counterstain with hematoxylin (blue) for improved visualization of tissue architecture

• Expected Results:

  • COL23A1 typically shows specific cytoplasmic localization in positive samples

  • Cell membrane localization may also be observed in some samples

How should dilution and optimization be approached for different COL23A1 antibody applications?

Recommended dilutions vary by application and antibody source:

For monoclonal antibodies, concentration-based dosing (μg/mL) is preferable to dilution ratios:

• WB: 2 μg/mL

• IHC: 25 μg/mL

Critical Note: All antibodies should be titrated in each testing system to obtain optimal results as sample type and experimental conditions can significantly impact performance .

How is COL23A1 implicated in viral pathogenesis, particularly in HSV-1 infection?

Recent research has revealed a novel role for COL23A1 in viral pathogenesis:

• Enhanced Viral Susceptibility:

  • Overexpression of COL23A1 in human keratinocytes (HaCaT cells) increases susceptibility to HSV-1 infection by approximately 1.6-fold

  • COL23A1 enhances both initial HSV-1 infection and subsequent cell-to-cell viral spread

  • Primary keratinocytes from patients with higher COL23A1 expression show increased HSV-1 susceptibility

• Molecular Mechanism:

  • Transcriptomic analysis suggests COL23A1 overexpression dampens keratinocyte immune responses

  • The mechanism appears similar to MARCO (macrophage scavenger receptor), which enhances HSV-1 binding to host cells

  • COL23A1 may facilitate viral entry and spread through altered cell-cell adhesion properties

• Clinical Relevance:

  • A single nucleotide polymorphism (SNP rs2973744) in COL23A1 has been identified as a risk factor for eczema herpeticum (EH) in atopic dermatitis patients

  • This SNP was found in 5% of ADEH+ patients (atopic dermatitis with eczema herpeticum), 1.6% of healthy donors, and 0% of ADEH- patients (atopic dermatitis without eczema herpeticum)

What is the significance of COL23A1 in cancer research and how are antibodies used in this context?

COL23A1 has demonstrated important roles in cancer progression:

• Expression Pattern:

  • COL23A1 is upregulated in many transformed cell lines

  • Serves as an indicator for tumor aggressiveness in prostate cancer

  • Has been studied in clear cell renal cell carcinoma as having oncogenic potential

• Functional Significance:

  • Knockdown of COL23A1 reduces cell adhesion and impairs anchorage-independent growth

  • Overexpression enhances cell adhesion in cancer cell lines

  • Promotes metastasis by aiding cell-cell and cell-matrix interactions

• Antibody Applications in Cancer Research:

  • Immunohistochemistry of adenocarcinoma samples shows specific cytoplasmic localization

  • Western blotting detects COL23A1 in human lung carcinoma cell lines

  • Multiple antibodies validated for cancer tissue samples enable reliable detection in various tumor types

How might genetic variants in COL23A1 influence protein function and disease susceptibility?

The relationship between COL23A1 genetic variants and disease has emerged as an important research area:

• SNP rs2973744 and Eczema Herpeticum:

  • This heterozygous SNP results in a differentially processed form of COL23A1

  • Leads to higher transcript and total protein levels in the presence of other risk factors

  • Associated with increased susceptibility to HSV-1 infection and development of eczema herpeticum

• Molecular Consequences:

  • Similar to mechanisms observed in hereditary thrombocythaemia, where mutations lead to more efficient translation of mutated transcripts

  • Results in altered protein structure and expression levels that modify host-pathogen interactions

  • May impact extracellular matrix organization and cell adhesion properties

• Implications for Antibody Selection:

  • When studying genetic variants, consider antibodies that recognize regions outside the variant location

  • For detecting specific variant forms, antibodies targeting the variant region may be required

  • Validation with samples of known genotype is critical for accurate interpretation

Why do observed molecular weights of COL23A1 differ from theoretical calculations?

The discrepancy between calculated (52 kDa) and observed (68-75 kDa) molecular weights is a common consideration when working with COL23A1 antibodies:

• Post-translational Modifications:

  • Glycosylation and other modifications increase apparent molecular weight

  • Different cell/tissue types may produce COL23A1 with varying modification patterns

  • COL23A1 has been observed at both 75 kDa and 60 kDa in some samples

• Technical Considerations:

  • Separation conditions (reducing vs. non-reducing) affect protein migration

  • The presence of fusion tags in recombinant proteins (e.g., GST tag adds approximately 26 kDa)

  • Protein cleavage has been described as a post-translational modification

• Validation Approach:

  • Use cell lines or tissues with known COL23A1 expression as positive controls

  • Mouse thymus tissue and NCI-H460 human lung carcinoma cells are validated positive controls

  • Multiple antibodies targeting different epitopes can confirm specificity

What strategies can help overcome inconsistent results with COL23A1 antibodies?

When facing inconsistent results:

• Antibody Selection Considerations:

  • Epitope location: Different antibodies target different regions (N-terminal AA 1-309, internal region AA 338-410, C-terminal)

  • Clonality: Monoclonal antibodies provide higher specificity but may be sensitive to epitope modifications

  • Host species: Consider secondary antibody compatibility and potential cross-reactivity

• Protocol Optimization:

  • Antigen retrieval: For IHC, compare Tris-EDTA buffer (pH 9.0) versus citrate buffer (pH 6.0)

  • Dilution optimization: Test a range of dilutions around the recommended value

  • Incubation conditions: Extended incubation at 4°C may improve signal for some applications

• Sample-Specific Adjustments:

  • Different tissue types may require specific processing methods

  • Cancer vs. normal tissue may show different expression patterns

  • Consider genetic variants that may affect epitope recognition

How can researchers differentiate between specific and non-specific signals when using COL23A1 antibodies?

To distinguish specific from non-specific signals:

• Essential Controls:

  • Positive control: Mouse thymus tissue for WB; human colon tissue or adenocarcinoma for IHC

  • Negative control: Omit primary antibody while maintaining all other conditions

  • Competing peptide/antigen: Pre-incubation with immunizing peptide should abolish specific signal

• Expected Patterns:

  • Western Blot: Primary bands at 68-75 kDa, with possible secondary band at 60 kDa in some samples

  • IHC/IF: Primarily cytoplasmic localization in positive samples

  • Cellular distribution should be consistent with membrane and cytoplasmic localization

• Advanced Validation Approaches:

  • siRNA/shRNA knockdown: Reduced signal following COL23A1 knockdown confirms specificity

  • Recombinant expression: Overexpression should increase signal intensity

  • Multiple antibodies: Concordant results with antibodies targeting different epitopes strongly supports specificity