CHST14 Antibody, Biotin conjugated

What is CHST14 and what is its biological significance?

CHST14 (Carbohydrate N-Acetylgalactosamine 4-0 Sulfotransferase 14), also known as D4ST1, is an enzyme that transfers sulfate to the C-4 hydroxyl of N-acetylgalactosamine residues in dermatan sulfate . This enzyme plays a critical role in the biosynthesis of dermatan sulfate, a glycosaminoglycan component of the extracellular matrix.

The biological significance of CHST14 is highlighted by its association with Ehlers-Danlos syndrome, musculocontractural type 1 (mcEDS-CHST14) . Mutations in the CHST14 gene disrupt proper dermatan sulfate formation, which affects collagen fibril assembly and extracellular matrix integrity. Transmission electron microscopy of skin specimens from mcEDS-CHST14 patients shows dispersed collagen fibrils instead of the tightly assembled fibrils observed in healthy controls .

What are the key specifications of commercially available CHST14 antibodies with biotin conjugation?

Commercial CHST14 antibodies with biotin conjugation typically recognize specific amino acid sequences within the CHST14 protein. For example, one commercially available antibody targets amino acids 70-133 of human CHST14 . Key specifications include:

• Target specificity: Human CHST14 (amino acids 70-133)

• Host: Rabbit

• Clonality: Polyclonal

• Conjugate: Biotin

• Applications: ELISA, Western Blotting (WB), Immunohistochemistry (IHC)

• Reactivity: Human

• Purification method: Protein G purified (>95% purity)

• Immunogen: Recombinant Human Carbohydrate sulfotransferase 14 protein (amino acids 70-133)

These specifications are important for researchers to consider when selecting the appropriate antibody for their experimental needs.

What applications are most suitable for biotin-conjugated CHST14 antibodies?

Biotin-conjugated CHST14 antibodies are particularly valuable for several research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): The biotin conjugation allows for signal amplification through the strong biotin-streptavidin interaction, enhancing detection sensitivity .

• Immunohistochemistry (IHC): Biotin-conjugated antibodies can be used in conjunction with streptavidin-enzyme complexes to visualize CHST14 expression in tissue sections .

• Western Blotting: Though biotin conjugation is less commonly used for this application, it can provide enhanced sensitivity when used with streptavidin-HRP detection systems .

How should researchers properly store and handle biotin-conjugated CHST14 antibodies?

Proper storage and handling of biotin-conjugated CHST14 antibodies are essential for maintaining antibody integrity and experimental reproducibility:

• Storage temperature: Store at -20°C for long-term storage and at 2-8°C for short-term storage (typically less than one month).

• Aliquoting: Upon first use, divide the antibody into small aliquots to minimize freeze-thaw cycles, which can degrade the antibody and reduce its effectiveness.

• Buffer conditions: Store in buffers containing stabilizers such as bovine serum albumin (BSA) or glycerol.

• Light exposure: Minimize exposure to light, especially if the biotin is coupled with fluorescent molecules.

• Working dilution preparation: When preparing working dilutions, use fresh buffer solutions and store diluted antibodies at 4°C, preferably for no longer than one week.

• Contamination prevention: Use sterile techniques to prevent microbial contamination, which can degrade the antibody.

Following these guidelines helps ensure optimal antibody performance and extends the useful life of the reagent.

How does biotin conjugation affect CHST14 antibody performance and what strategies can prevent biotin interference?

Biotin conjugation enhances detection sensitivity but can introduce specific challenges that researchers need to address:

Effects on antibody performance:

• Enhanced signal amplification: The biotin-streptavidin interaction provides significant signal amplification, improving detection sensitivity.

• Potential epitope masking: The biotin moiety may occasionally mask epitopes or alter antibody binding characteristics.

• Interference susceptibility: Biotin-conjugated antibodies are susceptible to interference from endogenous or exogenous biotin in samples .

Strategies to prevent biotin interference:

• Increased streptavidin concentration: Using excess streptavidin-coated magnetic microparticles (M) can neutralize free biotin in samples. Studies have demonstrated that higher concentrations of streptavidin-coated particles significantly improved resistance to biotin interference .

• Sample pretreatment: For samples potentially containing high biotin levels (e.g., from patients taking biotin supplements), pretreatment with streptavidin to absorb free biotin can reduce interference.

• Alternative detection systems: In cases of persistent biotin interference, consider using non-biotin detection systems or alternative conjugates like horseradish peroxidase (HRP) or fluorescent dyes directly linked to the antibody.

• Sample dilution: Diluting samples can reduce biotin concentration below interference thresholds while maintaining adequate antigen levels for detection.

A study evaluating anti-biotin interference methods demonstrated that increasing streptavidin-coated magnetic microparticle concentration effectively neutralized biotin in samples, maintaining assay accuracy even with biotin concentrations up to 5,000-10,000 ng/mL .

How can CHST14 antibodies be used to investigate the pathophysiology of Ehlers-Danlos syndrome?

CHST14 antibodies, including biotin-conjugated variants, are valuable tools for investigating the molecular pathophysiology of Ehlers-Danlos syndrome, musculocontractural type 1 (mcEDS-CHST14):

• Protein expression analysis: CHST14 antibodies can assess protein expression levels in patient-derived samples using western blotting or immunohistochemistry, helping correlate genotype with protein expression patterns.

• Tissue distribution studies: Immunohistochemical analysis using CHST14 antibodies allows visualization of CHST14 distribution in various tissues. Similar approaches have been used with decorin (a proteoglycan modified by CHST14) to examine extracellular matrix composition in Chst14 knockout mouse models .

• Functional assays: CHST14 antibodies can be used in activity inhibition studies to assess the functional consequences of specific mutations.

• Protein-protein interaction studies: Co-immunoprecipitation using CHST14 antibodies can identify interaction partners in normal versus disease states.

• Therapeutic development: CHST14 antibodies can monitor protein expression in response to potential therapeutic interventions targeting the dermatan sulfate biosynthetic pathway.

In mouse models of mcEDS-CHST14, immunohistochemical analysis has revealed altered decorin expression patterns in skin samples from Chst14-deficient mice compared to wild-type counterparts . Such studies provide insights into how CHST14 deficiency affects extracellular matrix composition.

What are the critical considerations when validating CHST14 antibody specificity for research applications?

Validating antibody specificity is crucial for ensuring reliable and reproducible research results. For CHST14 antibodies, consider the following validation approaches:

• Genetic validation: Testing the antibody in tissues or cells with confirmed CHST14 knockout or knockdown. The antibody signal should be absent or significantly reduced in these samples. Studies with Chst14 knockout mice have demonstrated this approach for validating CHST14-related reagents .

• Western blot analysis: Verifying that the antibody detects a band of the expected molecular weight (approximately 37 kDa for CHST14). Multiple bands may indicate cross-reactivity or protein processing.

• Peptide competition: Pre-incubating the antibody with the immunizing peptide (e.g., amino acids 70-133 for certain CHST14 antibodies) should abolish specific staining .

• Cross-species reactivity assessment: Testing the antibody against CHST14 from different species to confirm specificity when conducting comparative studies.

• Correlation with mRNA expression: Comparing antibody staining patterns with mRNA expression data from techniques like RT-PCR or RNA-seq. In Chst14 mutant mouse studies, quantification of Chst14 mRNA has been correlated with protein detection to validate findings .

• Mass spectrometry validation: Using immunoprecipitation followed by mass spectrometry to confirm that the antibody captures the intended target.

Implementing these validation steps ensures reliable antibody performance and strengthens the scientific validity of research findings.

How do different detection methods compare when using biotin-conjugated CHST14 antibodies in complex tissue samples?

When working with complex tissue samples, various detection methods can be employed with biotin-conjugated CHST14 antibodies, each with distinct advantages and limitations:

For immunohistochemical detection of CHST14 in dermis samples, DAB (diaminobenzidine) labeling with horseradish peroxidase has been successfully employed in research studying decorin expression patterns in relation to CHST14 function . This approach provides good sensitivity while maintaining morphological context in tissue sections.

When selecting a detection method, researchers should consider:

• The abundance of CHST14 in their sample

• The need for colocalization with other markers

• The required resolution (cellular vs. subcellular)

• Potential endogenous biotin levels in the tissue type

• Available imaging equipment

What methodological approaches can resolve data inconsistencies when comparing CHST14 antibody results with genetic and biochemical analyses?

When researchers encounter discrepancies between CHST14 antibody results and other analytical approaches, several methodological strategies can help resolve these inconsistencies:

• Multiple antibody validation: Use multiple CHST14 antibodies targeting different epitopes (e.g., AA 70-133, AA 178-205, AA 231-280) to verify findings . Consistent results across different antibodies increase confidence in observations.

• Complementary biochemical assays: Integrate enzymatic activity assays that measure CHST14 sulfotransferase function. In studies of Chst14 mutant mice, HPLC analysis of chondroitin sulfate (CS) and dermatan sulfate (DS) disaccharides in skeletal muscle and urine samples provided biochemical verification of genetic alterations .

• Correlation with functional outcomes: Assess biological consequences of CHST14 alterations. For example, in Chst14-deficient mice, the absence of dermatan sulfate disaccharides (detected by HPLC analysis) correlated with increased chondroitin sulfate levels, confirming the functional impact of genetic manipulation .

• Quantitative mRNA analysis: Compare antibody-detected protein levels with mRNA expression using techniques like RT-PCR. In one study, quantification of Chst14 mRNA in cardiac muscle from wild-type, heterozygous, and homozygous mutant mice helped validate the functional consequences of gene editing .

• Tissue-specific considerations: Account for tissue-specific post-translational modifications or protein interactions that might affect antibody binding. Different tissues may show variable correlation between genetic status and antibody detection.

• Technical controls: Include appropriate positive and negative controls, including recombinant CHST14 protein, wild-type tissues, and known CHST14-deficient samples to calibrate assay performance.

When analyzed together, these complementary approaches provide a more complete picture of CHST14 status and function, helping to resolve apparent discrepancies between different analytical methods.