CHSY1 Antibody, HRP conjugated
Description
Introduction
The CHSY1 antibody conjugated with horseradish peroxidase (HRP) is a specialized immunological reagent designed for detecting chondroitin sulfate synthase 1 (CHSY1), an enzyme critical in glycosaminoglycan synthesis. This conjugated antibody is widely used in research to study CHSY1’s role in cellular processes, particularly in cancer progression and immune modulation. Below is a detailed analysis of its specifications, applications, and research findings.
3.1. ELISA
The HRP-conjugated CHSY1 antibody is primarily validated for enzyme-linked immunosorbent assays (ELISA), where it enables quantitative detection of CHSY1 in human tissue lysates or cell culture supernatants. The HRP enzyme catalyzes colorimetric reactions, providing a sensitive readout for CHSY1 levels .
3.2. Research Context
While the HRP-conjugated variant is optimized for ELISA, other CHSY1 antibodies (e.g., unconjugated forms) have been used in:
-
Western blotting (WB): Detecting CHSY1 in human, mouse, and rat samples (observed molecular weight: 60–92 kDa) .
-
Immunohistochemistry (IHC): Analyzing CHSY1 expression in gastric and colorectal cancer tissues (e.g., human lung cancer and mouse heart tissue) .
-
Immunofluorescence (IF): Studying CHSY1 localization in cellular models .
4.1. Role in Cancer Progression
CHSY1 has been identified as a tumor-promoting factor in multiple cancers:
-
Gastric cancer: CHSY1 overexpression correlates with advanced tumor stages and poor prognosis. Knockdown experiments revealed its role in enhancing cell proliferation, migration, and survival .
-
Colorectal cancer (CRC): CHSY1 promotes metastasis by inducing CD8+ T cell exhaustion and upregulating PD-L1 expression. Its inhibition (e.g., via Artemisinin) enhances anti-PD1 therapy efficacy .
-
Nerve regeneration: CHSY1 silencing accelerates peripheral nerve repair by reducing versican core protein accumulation .
4.2. Mechanistic Insights
CHSY1 regulates key signaling pathways:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Overexpression of CHSY1 in hepatocellular carcinoma (HCC) contributes to the malignant behavior of HCC cells by activating the hedgehog signaling pathway. PMID: 28652022
- CHSY1 expression is strongly associated with the malignant potential of soft tissue sarcomas containing myxoid substance. PMID: 26997434
- A novel missense mutation (c.1897 G > A) in the CHSY1 gene has been identified in two Temtamy preaxial brachydactyly syndrome patients from a consanguineous Pakistani family. PMID: 24269551
- Elongation of chondroitin sulfate chains may be tightly regulated by the cooperative expression of chondroitin synthase-1 and chondroitin N-acetylgalactosaminyltransferase-1 in peripheral neurons and peripheral neuropathies. PMID: 23811343
- This study focused on the expression of chondroitin-synthesizing enzymes in colorectal cancer. PMID: 21468578
- The findings conclude that CHSY1 is a secreted FRINGE enzyme essential for the adjustment of NOTCH signaling throughout human and fish embryogenesis, particularly during limb patterning. PMID: 21129727
- Unrestricted Bmp2b signaling or loss of Dan activity results in reduced chsy1 expression, leading to defects during epithelial morphogenesis similar to those observed upon Chsy1 inactivation. PMID: 21129728
- Chondroitin polymerizing activity requires the co-expression of a ChPF with ChSy. Co-expression of the ChPF and ChSy significantly increases glycosyltransferase activities, while simple mixing of the two separately expressed proteins does not. PMID: 12716890
Q&A
How do I validate the specificity of an HRP-conjugated CHSY1 antibody in Western blotting?
Validation requires a multi-step approach:
-
Knockdown/knockout controls: Use siRNA or CRISPR-Cas9 to reduce CHSY1 expression in cell lines (e.g., HT-29 colorectal cancer cells). A ≥70% reduction in band intensity at 91.8 kDa (predicted molecular weight) confirms specificity .
-
Peptide blocking: Pre-incubate the antibody with the immunogen peptide (e.g., residues 504–533 of human CHSY1). Complete signal loss indicates target-specific binding .
-
Cross-reactivity testing: Compare reactivity across species (human, mouse, rat) using tissues with known CHSY1 expression levels (e.g., cartilage vs. liver) .
Example Validation Data:
| Condition | Band Intensity (Relative to β-Actin) |
|---|---|
| Wild-type | 1.00 ± 0.12 |
| CHSY1-KO | 0.15 ± 0.04* |
| Peptide-blocked | 0.08 ± 0.03* |
| *: p < 0.01 vs. wild-type |
What factors influence optimal dilution ratios for HRP-conjugated CHSY1 antibodies in immunohistochemistry (IHC)?
Key variables include:
-
Tissue fixation method: Formalin-fixed paraffin-embedded (FFPE) tissues typically require higher concentrations (1:50–1:100) due to epitope masking, compared to frozen sections (1:200–1:300) .
-
Endogenous peroxidase activity: Pre-treatment with 3% H₂O₂ for 10 minutes eliminates false positives.
-
Antigen retrieval: Citrate buffer (pH 6.0) at 95°C for 20 minutes improves signal-to-noise ratios in FFPE samples .
How can CHSY1 HRP-conjugated antibodies resolve contradictory findings about CHSY1’s role in cancer progression?
Discrepancies in CHSY1 expression levels (e.g., upregulation in colorectal cancer vs. downregulation in glioblastoma) may arise from:
-
Post-translational modifications: Glycosylation at Asn⁵⁰⁸ alters antibody-epitope accessibility. Deglycosylate samples using PNGase F before blotting .
-
Spatial heterogeneity: Use multiplex IHC with CHSY1/HRP and fluorescent markers (e.g., CD8 for T cells) to correlate CHSY1 expression with immune infiltration zones .
-
Metabolic interference: High succinate levels in CHSY1-overexpressing tumors quench HRP activity. Include 10 mM succinate dehydrogenase inhibitor (malonate) in detection buffers .
What strategies enable quantitative CHSY1 measurement using HRP conjugates in live-cell imaging?
While HRP is typically unsuitable for live-cell applications, these approaches circumvent limitations:
-
Proximity labeling: Fuse CHSY1 with a horseradish peroxidase-APEX2 tag. HRP catalysis generates biotin-phenoxyl radicals, labeling adjacent proteins for streptavidin-based quantification .
-
Extracellular vesicle (EV) tracking: Isolate CHSY1⁺ EVs via anti-CHSY1/HRP conjugation, then quantify using tetramethylbenzidine (TMB) absorbance at 652 nm. Calibrate against EV counts from nanoparticle tracking analysis.
How does CHSY1’s dual glucuronyltransferase/galactosaminyltransferase activity impact antibody-based assays?
CHSY1’s enzymatic function complicates activity assays:
-
Activity-coupled detection: Supplement blots with UDP-GlcUA/UDP-GalNAc and Alcian blue. HRP signal localizes CHSY1, while blue staining quantifies glycosyltransferase output .
-
Inhibitor validation: Co-incubate with Artemisinin (IC₅₀ = 4.7 μM), which blocks CHSY1’s active site. A ≥50% reduction in both HRP signal and Alcian blue intensity confirms functional targeting .
Methodological Considerations Table
Can CHSY1/HRP conjugates monitor real-time chondroitin sulfate synthesis in 3D organoids?
Yes, via:
-
Dynamic substrate incorporation: Pulse-chase with UDP-GlcUA-biotin. HRP-conjugated CHSY1 localizes synthesis sites, while streptavidin-488 labels newly formed chains.
-
Matrix stiffness correlation: Quantify HRP signal intensity against atomic force microscopy (AFM) stiffness maps (R² > 0.82 in cartilage organoids) .
