chst14 Antibody

What is CHST14 and what is its biological function?

CHST14, full name carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 14, is a critical enzyme for the biosynthesis of dermatan sulfate (DS) in vivo. It specifically catalyzes the transfer of a sulfate group from the sulfate donor 3′-phosphoadenosine 5′-phosphosulfate to the C-4 position of the GalNAc residue in DS chains . This post-translational modification is essential for the proper function of proteoglycans that regulate collagen fibrils through their interaction. CHST14 has a calculated molecular weight of 376 amino acids (43 kDa), although it typically appears at 45-50 kDa in experimental conditions . Mutations in the CHST14 gene cause musculocontractural type Ehlers-Danlos syndrome (mcEDS-CHST14), characterized by joint and skin hyperextensibility and fragility of various organs .

What applications are CHST14 antibody suitable for?

The CHST14 antibody (such as the 17749-1-AP polyclonal antibody) has been validated for multiple research applications:

It is recommended that researchers titrate this reagent in their specific testing systems to obtain optimal results, as the optimal dilution can be sample-dependent . When using this antibody for research requiring high specificity, validation experiments should be conducted for your specific tissue or cell type.

What is the species reactivity profile of CHST14 antibody?

The CHST14 antibody has been tested and validated to react with samples from multiple species:

While the antibody has been cited primarily in human research applications, the cross-reactivity with mouse and rat samples makes it valuable for comparative studies and animal models of disease . This cross-species reactivity is particularly useful when studying CHST14 in knockout mouse models as described in research on placental development and Ehlers-Danlos syndrome .

How can CHST14 antibody be used in cancer research protocols?

CHST14 has been implicated in cancer progression, particularly in gastric cancer where it demonstrates elevated expression. Research protocols using CHST14 antibody for cancer studies typically include:

• Expression analysis: CHST14 antibody (17749-1-AP at 1:1000 dilution) has been used to analyze protein expression levels in cancer cells versus normal tissue through immunoblotting techniques .

• Functional studies: After CHST14 knockdown using siRNA (sense: GCAGGCGACGAUGUCACAUTT, antisense: AUGUGACAUCGUCGCCUGCTT), the antibody can detect decreased protein expression, confirming successful knockdown before proceeding with functional assays .

• Pathway analysis: CHST14 antibody has been used in conjunction with antibodies against downstream markers like β-catenin, C-myc, CyclinD1, Snail, MMP2, and MMP9 to elucidate signaling pathways affected by CHST14 manipulation .

• Immunohistochemistry in tumors: IHC protocols using CHST14 antibody can evaluate expression patterns in tumor tissues, with scoring based on both staining intensity and percentage of positive cells .

A comprehensive cancer research approach would combine CHST14 protein detection with RT-qPCR using primers such as: 5ʹ-TACCACCTGTGCCAGCCTTGT-3ʹ and 5ʹ-GAAATCGGACGTGAGGTGGTG-3ʹ for expression validation at both protein and mRNA levels .

What methodologies are recommended for investigating CHST14's role in dermatan sulfate biosynthesis?

Investigating CHST14's role in dermatan sulfate biosynthesis requires multiple complementary approaches:

• Genetic models: Utilize Chst14 gene-deleted models (Chst14−/−) to study the functional consequences of CHST14 deficiency. These models can reveal critical information about DS biosynthesis and its biological significance .

• Glycosaminoglycan analysis: High-performance liquid chromatography (HPLC) can be employed to analyze disaccharide composition after enzymatic digestion with chondroitinase B (for DS moiety) or chondroitinase AC (for CS moiety) .

• Quantitative comparison: Compare DS and CS disaccharide content between wild-type, heterozygous, and homozygous CHST14 knockout samples. The CHST14−/− samples typically show significantly decreased DS disaccharides while CS disaccharides remain relatively unchanged .

Sample data from placental tissue analysis shows these differences:

These differences reflect CHST14's specific role in dermatan sulfate synthesis rather than chondroitin sulfate production .

How should researchers design experiments to investigate CHST14 in Ehlers-Danlos syndrome models?

When investigating CHST14 in Ehlers-Danlos syndrome research, consider these experimental design principles:

• Model selection: Although Chst14−/− mice were developed as potential models for mcEDS-CHST14, the high perinatal lethality limits their use as adult models. Therefore, placental analysis or conditional knockout approaches may be more practical .

• Vascular abnormality assessment: Since large subcutaneous hematomas are a serious complication in mcEDS-CHST14, focus on vascular structure analysis using:

  • Histological examination for alterations in vascular structure

  • Assessment of ischemic and/or necrotic-like changes

  • Electron microscopy to evaluate basement membrane structure of capillaries

• Collagen structure analysis: Evaluate the impact of DS deficiency on collagen fibril organization using electron microscopy and mechanical property tests, as DS is involved in regulating collagen fibrils through proteoglycan interactions .

• Proteoglycan analysis: Analyze changes in proteoglycans that contain DS side chains, particularly biglycan, which directly interacts with collagen fibrils .

• Immunohistochemical staining: Use CHST14 antibody at 1:20-1:200 dilution with proper antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) to assess protein expression in relevant tissues .

How can researchers optimize Western blot protocols for CHST14 antibody?

To achieve optimal Western blot results with CHST14 antibody, follow these methodological considerations:

• Sample preparation:

  • For cell lines: HEK-293 and HepG2 cells have shown positive results and can serve as positive controls

  • Use RIPA buffer supplemented with protease inhibitors for protein extraction

  • Denature samples at 95°C for 5 minutes in loading buffer containing SDS and β-mercaptoethanol

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels for optimal separation around the 45-50 kDa range

  • Transfer to nitrocellulose membranes at 100V for 60-90 minutes in cold transfer buffer

• Antibody incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Incubate with CHST14 antibody at 1:500-1:1000 dilution overnight at 4°C

  • Wash three times with TBST

  • Incubate with appropriate secondary antibody for 1 hour at room temperature

• Detection and troubleshooting:

  • For weak signals: Extend primary antibody incubation time or increase concentration

  • For high background: Increase blocking time or use alternative blocking reagents

  • For multiple bands: Validate specificity with positive and negative controls

  • Use GAPDH (1:15,000; #60,004–1-Ig) as a loading control

What are the key considerations for optimizing immunohistochemistry with CHST14 antibody?

For effective immunohistochemical detection of CHST14, consider these methodological approaches:

• Tissue preparation and antigen retrieval:

  • Use paraffin-embedded tissue sections for consistent results

  • Recommended antigen retrieval: TE buffer pH 9.0 (primary recommendation)

  • Alternative antigen retrieval: citrate buffer pH 6.0

• Antibody dilution optimization:

  • Initial recommended range: 1:20-1:200

  • Perform titration experiments to determine optimal concentration for your specific tissue

  • Positive control tissues: human placenta, brain, kidney, and spleen

• Detection system selection:

  • Use detection systems appropriate for rabbit IgG antibodies

  • Consider amplification systems for low-abundance targets

• Result evaluation:

  • Use scoring index (SI) by multiplying the percentage of positive cells and staining intensity

  • Have two independent pathologists evaluate samples in a blinded manner

  • Document staining patterns with representative images at multiple magnifications

What controls should be included when validating CHST14 antibody specificity?

Proper validation of CHST14 antibody specificity requires systematic inclusion of controls:

• Positive controls:

  • Cell lines: HEK-293 and HepG2 cells have demonstrated positive Western blot results

  • Tissues: Human placenta, brain, kidney, and spleen have shown positive IHC results

  • Recombinant CHST14 protein or overexpression lysates if available

• Negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • CHST14 knockdown samples: Cells transfected with CHST14 siRNA (sequence: sense: GCAGGCGACGAUGUCACAUTT, antisense: AUGUGACAUCGUCGCCUGCTT)

  • Tissue from Chst14−/− animal models, noting significant reduction in signal

• Specificity validation approaches:

  • Peptide competition assay using the immunogen (CHST14 fusion protein Ag12014)

  • Side-by-side comparison with alternative CHST14 antibodies targeting different epitopes

  • Correlation of protein detection with mRNA levels using RT-qPCR with validated primers

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with related sulfotransferases, particularly CHST11

  • Compare expression patterns with gene expression databases like TCGA and GEO

How can CHST14 antibody be incorporated into functional studies of cell migration and invasion?

CHST14 has been implicated in cancer cell migration and invasion, making these functional assays particularly relevant:

• Migration assay protocol:

  • First, perform CHST14 knockdown using siRNA transfection (48h duration)

  • Confirm knockdown efficiency by Western blot using CHST14 antibody (1:1000 dilution)

  • Seed cells in migration chambers with serum-free medium (200μL)

  • After appropriate incubation time (typically 48h), fix cells with methanol for 30 minutes

  • Stain with crystal violet and quantify migrated cells

• Invasion assay protocol:

  • Prepare Matrigel by diluting with BSA solution (10 g/L) at an 8:1 proportion

  • Add 40μL of dissolved Matrigel to each chamber

  • After 30 minutes, hydrate the Matrigel with 50μL BSA solution

  • Add cells in 200μL serum-free medium

  • Incubate for 48h, then fix, stain and quantify as in migration assay

• Data analysis considerations:

  • Perform at least three independent experiments

  • Use t-test for statistical analysis between two groups

  • For multi-group comparison, use one-way analysis of variance (ANOVA)

  • Consider p<0.05 as statistically significant

  • Document results with representative images and quantitative graphs

What approaches should be used to investigate CHST14's role in EMT and cancer progression?

To comprehensively investigate CHST14's role in epithelial-mesenchymal transition (EMT) and cancer progression:

• Protein expression analysis:

  • Use CHST14 antibody (1:1000 dilution) to assess baseline expression in various cancer cell lines

  • Compare with EMT markers using antibodies against:

    • β-catenin (1:1000; #51,067–2-AP)

    • C-myc (1:10,000; #67,447–1-Ig)

    • CyclinD1 (1:10,000; #60,186–1-Ig)

    • Snail (1:1000; #TA6032)

    • MMP2 (1:1000; #10,373–2-AP)

    • MMP9 (1:1000; #10,375–2-AP)

• Gene expression correlation:

  • Implement RT-qPCR using validated CHST14 primers (5ʹ-TACCACCTGTGCCAGCCTTGT-3ʹ and 5ʹ-GAAATCGGACGTGAGGTGGTG-3ʹ)

  • Correlate with EMT-related genes like β-catenin, c-Myc, and CyclinD1

  • Normalize using housekeeping genes like GAPDH

  • Calculate relative expression using the 2−ΔΔCt method

• Functional rescue experiments:

  • After CHST14 knockdown, introduce wild-type CHST14 to confirm phenotype rescue

  • Alternatively, introduce mutant CHST14 (similar to those found in mcEDS-CHST14 patients) to investigate mechanism

• In vivo validation:

  • Use CHST14 antibody for IHC in patient samples to correlate expression with clinical outcomes

  • Analyze CHST14 expression in relation to tumor stage, grade, and patient survival

  • Consider tissue microarray approach for high-throughput analysis

How should researchers interpret contradictory results in CHST14 expression studies?

When faced with contradictory results in CHST14 expression studies, consider these analytical approaches:

• Technical considerations:

  • Antibody specificity: Ensure the CHST14 antibody (17749-1-AP) has been properly validated

  • Detection method sensitivity: Western blot may show different results than IHC due to different sensitivities

  • Sample preparation variations: Different fixation methods can affect epitope accessibility

• Biological considerations:

  • Tissue-specific expression: CHST14 expression varies across tissues; placenta, brain, kidney, and spleen show positive results

  • Disease state variations: CHST14 may be upregulated in certain pathological conditions like gastric cancer

  • Developmental stage differences: Expression in Chst14+/+, Chst14+/−, and Chst14−/− models shows significant variation

• Data reconciliation approaches:

  • Multi-method validation: Combine protein detection (Western blot, IHC) with mRNA analysis (RT-qPCR)

  • Quantitative assessment: Use scoring systems for IHC and normalized band intensity for Western blots

  • Functional validation: Correlate expression levels with relevant functional outcomes (e.g., DS synthesis capacity)

  • Meta-analysis: Compare your results with published data from TCGA and GEO databases

• Statistical considerations:

  • Ensure appropriate statistical tests are applied (t-test for two groups, ANOVA for multiple groups)

  • Consider sample size limitations and biological variability

  • Perform at least three independent replicates of experiments

What are promising research areas for CHST14 antibody applications beyond current applications?

Several emerging research directions could benefit from CHST14 antibody applications:

• Therapeutic target validation:

  • Use CHST14 antibody to evaluate target engagement in drug development studies

  • Monitor changes in CHST14 expression after treatment with potential therapeutic compounds

  • Validate CHST14 as a biomarker for disease progression or treatment response

• Developmental biology:

  • Investigate CHST14's role in embryonic development using IHC in developmental stage series

  • Study the temporal and spatial expression patterns during organogenesis

  • Explore CHST14's function in placental development, as studies show its importance in vascular structure

• Extracellular matrix biology:

  • Examine how CHST14 expression correlates with collagen organization and ECM structure

  • Use co-localization studies with CHST14 antibody and ECM components

  • Investigate the relationship between CHST14, dermatan sulfate production, and tissue biomechanics

• Expanded disease associations:

  • Beyond mcEDS-CHST14 and gastric cancer, explore CHST14's potential role in other disorders

  • Investigate vascular abnormalities in different pathological conditions

  • Consider CHST14's involvement in wound healing and tissue repair processes