CHS1A Antibody

What is CHST1 and what are its primary biological functions?

CHST1 (Carbohydrate Sulfotransferase 1), also known as KSGal6ST or KSST, is an enzyme that catalyzes the transfer of sulfate groups from 3′-phosphoadenosine 5′-phosphosulfate to specific galactose residues on keratan sulfate chains, particularly those adjacent to sulfated N-acetylglucosamine. The enzyme is predominantly expressed in the brain and skeletal muscle, where it localizes to the trans-Golgi network. CHST1 plays a crucial role in maintaining corneal transparency through its sulfation of keratan sulfate, and it also contributes to the generation of L-selectin ligands involved in cell adhesion and migration. Mutations in the CHST1 gene have been implicated in macular corneal dystrophy, highlighting its significance in ocular health .

How does CHST1 differ from CHSY1 in terms of function and research applications?

CHST1 and CHSY1 are distinct enzymes with different biological functions and research applications. CHST1 is primarily involved in the sulfation of keratan sulfate and is essential for corneal transparency and L-selectin ligand formation. In contrast, CHSY1 (Chondroitin Sulfate Synthase 1) is involved in chondroitin sulfate synthesis and has been implicated in cancer progression. Recent research has demonstrated that CHSY1 promotes CD8+ T cell exhaustion through activation of the succinate metabolic pathway, contributing to colorectal cancer (CRC) liver metastasis. CHSY1 is overexpressed in both primary CRC tissues and liver metastases, correlating with worse clinical prognosis . These functional differences necessitate different experimental approaches and interpretations when studying each protein.

What detection methods are compatible with CHST1 antibodies?

CHST1 antibodies, such as the mouse monoclonal IgG2b kappa light chain antibody (A-2), are versatile tools compatible with multiple detection methods. These include western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA). The antibody is available in both non-conjugated forms and various conjugated forms, including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates. This versatility allows researchers to select the most appropriate detection method for their specific experimental requirements .

How should I optimize antibody dilutions for Western blotting of CHST1 in different tissue samples?

For optimal Western blotting of CHST1 in different tissue samples, a tiered optimization approach is recommended. Begin with a dilution range of 1:500 to 1:2000 using the standard concentration (200 μg/ml) of CHST1 Antibody (A-2). Since CHST1 is predominantly expressed in brain and skeletal muscle, expect stronger signals from these tissues compared to others. When comparing expression across multiple tissues, standardize protein loading (25-40 μg total protein per lane) and use consistent transfer conditions. For brain and skeletal muscle samples, start at higher dilutions (1:1000 to 1:2000) to prevent signal saturation, while for tissues with expected lower expression, begin with 1:500 dilutions. Always include positive controls (brain or skeletal muscle lysates) and negative controls (tissues with minimal CHST1 expression). Additionally, confirm specificity by using secondary-only controls and validate results with alternative detection methods when possible .

What are the recommended fixation and permeabilization protocols for immunofluorescence studies using CHST1 antibodies?

For immunofluorescence studies targeting CHST1, which localizes primarily to the trans-Golgi network, the following optimized protocol is recommended: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature, followed by permeabilization with 0.2% Triton X-100 for 10 minutes. Since CHST1 is a Golgi-resident protein, consider using complementary markers like GM130 for colocalization studies. For improved visualization of the trans-Golgi compartment, a mild permeabilization with 0.1% saponin may preserve Golgi morphology better than Triton X-100. When studying brain or skeletal muscle tissue sections, extend fixation to 24 hours in 4% paraformaldehyde, followed by cryoprotection in 30% sucrose before sectioning. Use CHST1 Antibody (A-2) FITC conjugate at 1:100 dilution for direct detection, or use the unconjugated antibody at 1:100-1:200 dilution followed by a fluorophore-conjugated secondary antibody. Include appropriate controls for autofluorescence and counterstain with DAPI to visualize cell nuclei .

What considerations should be made when using CHSY1 antibodies in cancer research models?

When using CHSY1 antibodies in cancer research models, several important considerations must be addressed. First, select appropriate tumor models that express CHSY1, particularly colorectal cancer (CRC) models, as CHSY1 has been shown to be overexpressed in CRC primary tissues and liver metastases. When designing in vivo experiments, incorporate both orthotopic and metastatic models to fully assess CHSY1's role in cancer progression. Include measurements of CD8+ T cell status, as recent research shows CHSY1 promotes CD8+ T cell exhaustion through activation of succinate metabolism pathways. Consider combination studies with immune checkpoint inhibitors like anti-PD1, since CHSY1 has been shown to upregulate PD-L1 expression. For mechanistic studies, incorporate analysis of the PI3K/AKT/HIF1A pathway, which has been implicated in CHSY1-mediated effects. Additionally, include metabolomic analyses focusing on succinate metabolism, which appears to be a key mechanism by which CHSY1 influences immune evasion and metastasis .

What are common sources of background in CHST1 immunodetection and how can they be minimized?

Common sources of background in CHST1 immunodetection include non-specific antibody binding, endogenous peroxidase activity (for HRP-based detection), inadequate blocking, and cross-reactivity with other sulfotransferases. To minimize these issues, implement a comprehensive optimization strategy: Increase blocking time to 2 hours using 5% BSA or milk in TBS-T and extend washing steps to 4-5 times for 10 minutes each. For Western blotting, use freshly prepared buffers and consider adding 0.1% SDS to washing solutions to reduce non-specific binding. When performing immunofluorescence, include an autofluorescence quenching step using 0.1% sodium borohydride or commercial quenching reagents. Since CHST1 shares sequence homology with other sulfotransferases, validate specificity using knockout or knockdown controls. For immunoprecipitation assays, pre-clear lysates with protein A/G beads before adding the CHST1 antibody and use IP-optimized detergents like NP-40 or CHAPS at 0.5-1%. Finally, when using the HRP-conjugated CHST1 antibody, block endogenous peroxidase activity with 0.3% H₂O₂ treatment for 10-15 minutes prior to antibody incubation .

How can I confirm the specificity of CHST1 antibody reactivity in my experimental system?

To confirm the specificity of CHST1 antibody reactivity, implement a multi-layered validation approach. First, perform peptide competition assays using the immunizing peptide to selectively block specific binding sites. Second, include positive controls known to express CHST1 (brain or skeletal muscle lysates) and negative controls with minimal CHST1 expression or CHST1 knockout samples. Third, validate results across multiple detection techniques (Western blot, immunofluorescence, and ELISA) to ensure consistent patterns. Fourth, compare reactivity patterns with published literature, particularly focusing on the expected molecular weight (approximately 42-45 kDa for human CHST1) and subcellular localization (trans-Golgi network). Fifth, analyze antibody cross-reactivity with other sulfotransferases by expressing recombinant sulfotransferases and testing antibody binding. Sixth, employ siRNA knockdown of CHST1 to demonstrate reduced antibody signal correlating with reduced CHST1 expression. Finally, consider orthogonal validation using antibodies targeting different epitopes of CHST1 to confirm consistent detection patterns .

What strategies can be employed when dealing with weak CHST1 or CHSY1 antibody signals in biological samples?

When confronted with weak CHST1 or CHSY1 antibody signals, several evidence-based enhancement strategies can be implemented. For Western blotting, increase protein loading (50-75 μg) and reduce antibody dilution (to 1:250-1:500). Consider using enhanced chemiluminescence substrates with higher sensitivity or switch to alternative detection methods like near-infrared fluorescent detection systems. Incorporate signal amplification methods such as tyramide signal amplification for immunohistochemistry or immunofluorescence. Optimize epitope retrieval methods: for CHST1, use citrate buffer (pH 6.0) heated to 95°C for 20 minutes, while for CHSY1, test both citrate and EDTA-based retrieval solutions. Enrich target proteins through immunoprecipitation before detection or use concentrated tissue lysates from regions known to express higher levels of the target protein. For low-abundance samples, consider the HRP-conjugated antibody formats which eliminate the need for secondary antibody incubation and reduce background. Extend primary antibody incubation to overnight at 4°C to increase binding opportunity, and optimize detergent concentration in wash buffers to reduce stringency while maintaining specificity .

How can CHST1 antibodies be utilized in studying corneal disorders and potential therapeutic approaches?

CHST1 antibodies serve as valuable tools for investigating corneal disorders, particularly macular corneal dystrophy, which has been linked to CHST1 gene mutations. For comprehensive studies of corneal pathology, implement a multi-modal approach: First, use immunohistochemistry with CHST1 antibodies to map expression patterns in healthy versus diseased corneal tissues, paying specific attention to the epithelium, stroma, and endothelium. Second, combine CHST1 immunostaining with keratan sulfate detection to correlate enzyme expression with substrate sulfation patterns. Third, establish corneal epithelial and keratocyte cell cultures to investigate CHST1 expression and function under normal and stress conditions. Fourth, employ CHST1 antibodies to track protein expression changes in response to potential therapeutic compounds that might modulate keratan sulfate sulfation. Fifth, use co-immunoprecipitation with CHST1 antibodies to identify protein interaction partners that might represent alternative therapeutic targets. Finally, develop ex vivo corneal models where CHST1 expression can be manipulated, then measure changes in corneal transparency, hydration, and biomechanical properties. This comprehensive approach can provide insights into how CHST1 dysfunction contributes to corneal disorders and identify potential therapeutic strategies .

What are the methodological approaches for investigating the role of CHSY1 in cancer immune evasion?

To investigate CHSY1's role in cancer immune evasion, implement a comprehensive methodological framework. Begin with multiparameter flow cytometry to characterize CD8+ T cell populations in CHSY1-high versus CHSY1-low tumors, quantifying exhaustion markers (PD-1, LAG-3, TIM-3, TIGIT) and functional markers (IFN-γ, TNF-α, Granzyme B). Establish CHSY1 knockdown and overexpression models in cancer cell lines, then conduct co-culture experiments with CD8+ T cells to assess functional changes in T cell activation and tumor cell killing capacity. Perform metabolomic analysis focusing on succinate pathway metabolites using liquid chromatography-mass spectrometry to validate CHSY1's impact on metabolic reprogramming. Use ChIP-seq and RNA-seq to map CHSY1-dependent transcriptional changes in both tumor cells and infiltrating T cells. Implement spatially-resolved technologies like multiplexed immunofluorescence or imaging mass cytometry to characterize the tumor immune microenvironment in relation to CHSY1 expression patterns. Conduct in vivo studies with immune-competent mouse models, combining CHSY1 manipulation with immune checkpoint blockade therapy. Finally, isolate tumor-infiltrating lymphocytes from CHSY1-high versus CHSY1-low tumors for ex vivo functional assays to directly assess T cell exhaustion parameters .

How can antibody-based approaches be combined with genetic techniques to study CHST1/CHSY1 function?

Integrating antibody-based approaches with genetic techniques creates a powerful methodology for elucidating CHST1/CHSY1 function. Begin by using CRISPR/Cas9 to generate knockout or knockdown cell lines, then validate protein absence or reduction with specific antibodies via Western blotting and immunofluorescence. Implement antibody-based chromatin immunoprecipitation followed by sequencing (ChIP-seq) in conjunction with RNA-seq to identify both regulatory regions bound by transcription factors controlling CHST1/CHSY1 expression and the downstream transcriptional changes resulting from their modulation. For mechanistic studies, employ proximity ligation assays (PLA) with CHST1/CHSY1 antibodies paired with antibodies against suspected interaction partners to visualize protein-protein interactions in situ. Create domain-specific mutants of CHST1/CHSY1 and use conformation-specific antibodies to assess structural changes affecting function. For translational research, combine genetic screening of patient samples for CHST1/CHSY1 mutations with immunohistochemical analysis using specific antibodies to correlate genotype with protein expression patterns. Finally, implement genetic rescue experiments in knockout models followed by antibody-based detection methods to confirm functional restoration and specify essential domains or residues .

How should researchers interpret differences in antibody reactivity between CHST1 and its isoforms or related proteins?

When interpreting differences in antibody reactivity between CHST1 and its isoforms or related proteins, researchers should implement a structured analytical approach. First, confirm the specificity of the antibody by examining the epitope region—the CHST1 Antibody (A-2) recognizes a specific epitope that may be conserved or varied across isoforms. Second, analyze the expression patterns across tissues, as CHST1 is predominantly expressed in brain and skeletal muscle, whereas related sulfotransferases may show different tissue distributions. Third, examine subcellular localization data carefully; CHST1 localizes specifically to the trans-Golgi network, and any deviation may indicate cross-reactivity with related proteins. Fourth, compare molecular weights observed in Western blots—human CHST1 appears at approximately 42-45 kDa, while isoforms or related proteins may migrate differently. Fifth, conduct parallel experiments using antibodies targeting different epitopes of CHST1 or specific antibodies against related sulfotransferases to differentiate signals. Finally, validate findings with orthogonal methods such as mass spectrometry or RNA-seq to confirm protein identity when antibody cross-reactivity is suspected .

What are the implications of CHSY1 in cancer immunotherapy research, and how can antibodies help understand resistance mechanisms?

CHSY1's emerging role in cancer immunotherapy research has significant implications for understanding treatment resistance mechanisms. Recent findings indicate that CHSY1 promotes CD8+ T cell exhaustion through activation of the succinate metabolism pathway, leading to colorectal cancer liver metastasis. This process involves upregulation of PD-L1 expression, suggesting a direct mechanism of immune evasion. Anti-CHSY1 antibodies can be instrumental in mapping expression patterns in responders versus non-responders to immunotherapy, potentially identifying a biomarker for treatment stratification. The combination of CHSY1 knockdown with anti-PD1 therapy has demonstrated synergistic resistance to CRC liver metastasis, indicating a novel therapeutic approach. Artemisinin has been identified as a CHSY1 inhibitor that reduces liver metastasis and enhances anti-PD1 efficacy. Researchers can employ anti-CHSY1 antibodies in multiplexed immunohistochemistry to correlate CHSY1 expression with immune cell infiltration patterns and checkpoint molecule expression. Single-cell approaches combining antibody-based protein detection with transcriptomics can further elucidate cell-specific mechanisms of CHSY1-mediated immune evasion .

How can researchers address cross-reactivity concerns when studying antibody responses against glycosylated proteins like those modified by CHST1?

Addressing cross-reactivity concerns when studying antibody responses against glycosylated proteins requires a comprehensive technical approach. First, implement glycosidase treatments (such as PNGase F or O-glycosidase) to remove specific glycan modifications before antibody detection, allowing comparison of glycosylated versus deglycosylated signals. Second, use lectins specific for sulfated glycans in parallel with CHST1 antibodies to differentiate between protein recognition and glycan recognition. Third, generate recombinant proteins with site-directed mutagenesis at known glycosylation sites to create glycosylation-deficient variants for comparative studies. Fourth, employ competitive binding assays with free sulfated glycans to determine if antibody binding is inhibited, suggesting glycan-directed reactivity. Fifth, conduct epitope mapping using peptide arrays or hydrogen-deuterium exchange mass spectrometry to precisely identify the binding regions of antibodies. Sixth, for clinical samples, pre-adsorb sera with glycan-rich but target-protein-poor samples to deplete glycan-reactive antibodies. Finally, validate findings using multiple antibody clones recognizing different epitopes, ideally including at least one antibody targeting a non-glycosylated region of the protein .

Table 1: Comparative Analysis of CHST1 and CHSY1 Antibodies in Different Research Applications

What factors should be considered when selecting between polyclonal and monoclonal antibodies for CHST1/CHSY1 research?

When selecting between polyclonal and monoclonal antibodies for CHST1/CHSY1 research, researchers should consider several critical factors. Experimental objective is paramount—monoclonal antibodies like CHST1 Antibody (A-2) offer superior specificity and reproducibility for precise localization studies, quantitative analyses, and applications requiring batch consistency. Conversely, polyclonal antibodies provide improved sensitivity through recognition of multiple epitopes, which can be advantageous for detecting low-abundance CHST1/CHSY1 in certain tissues or when protein conformation may be altered. For denatured applications like Western blotting, both antibody types can be effective, though polyclonals may detect partially degraded proteins more efficiently. For native condition experiments like immunoprecipitation, consider epitope accessibility—the monoclonal CHST1 Antibody (A-2) recognizes a specific epitope that must remain accessible, while polyclonals offer multiple binding sites. Cross-reactivity risks differ significantly—monoclonals minimize cross-reaction with related sulfotransferases/synthases but may miss splice variants, while polyclonals detect more variants but with higher cross-reactivity risk. Finally, consider application-specific requirements: for therapeutic development or diagnostic applications, monoclonals provide necessary consistency; for initial characterization or challenging detection scenarios, polyclonals offer versatility .

How are recent findings about CHSY1's role in T cell exhaustion changing our understanding of cancer immunology?

Recent findings regarding CHSY1's role in T cell exhaustion represent a significant paradigm shift in cancer immunology understanding. CHSY1 has been identified as a key mediator promoting CD8+ T cell exhaustion through activation of the succinate metabolism pathway, directly contributing to colorectal cancer liver metastasis. This discovery establishes a novel mechanistic link between glycosaminoglycan synthesis enzymes and immune evasion—traditionally separate research domains. The research revealed that CHSY1 upregulates PD-L1 expression and activates the PI3K/AKT/HIF1A pathway, creating an immunosuppressive microenvironment favorable for cancer metastasis. Most significantly, CHSY1 knockdown demonstrated synergistic effects with anti-PD1 immunotherapy in reducing liver metastasis, suggesting that targeting CHSY1 could overcome resistance to immune checkpoint inhibitors. This finding challenges current immunotherapy approaches by identifying metabolic pathway modulation as a critical adjunct strategy. Moreover, the identification of artemisinin as a CHSY1 inhibitor that can enhance anti-PD1 efficacy provides a concrete translational pathway toward improving immunotherapy outcomes, particularly for metastatic colorectal cancer patients who currently have limited treatment options .

What methodological advances are needed to better study the functional interactions between CHST1/CHSY1 and their substrate proteins?

To better study functional interactions between CHST1/CHSY1 and their substrate proteins, several methodological advances are urgently needed. First, develop proximity-dependent labeling techniques optimized for Golgi-resident enzymes like CHSY1 and CHST1, using engineered peroxidases or ligases fused to these enzymes to identify proximal substrates in their native cellular context. Second, implement CRISPR-based screens with glycan-specific readouts to systematically identify proteins affected by CHST1/CHSY1 activity. Third, advance glycoproteomics methodologies specific for sulfated glycans, including improved enrichment strategies and mass spectrometry fragmentation techniques that preserve sulfate modifications during analysis. Fourth, develop real-time imaging approaches using fluorescent sulfate donors or acceptors to visualize enzyme-substrate interactions in living cells. Fifth, create synthetic biology platforms with engineered glycosyltransferases/sulfotransferases having expanded substrate specificities or orthogonal donor preferences. Sixth, establish cell-free reconstitution systems with purified components to directly study enzyme kinetics and substrate selectivity. Finally, develop computational models integrating structural data, reaction kinetics, and cellular localization to predict enzyme-substrate interactions and the effects of mutations or inhibitors on glycan synthesis pathways .

How might emerging antibody technologies enhance our ability to study CHST1/CHSY1 in complex biological systems?

Emerging antibody technologies are poised to revolutionize CHST1/CHSY1 research in complex biological systems. Next-generation bi-specific antibodies can simultaneously target CHST1/CHSY1 and their interaction partners or downstream signaling molecules, providing spatial insights into functional complexes. Antibody-based proximity assays like proximity extension assays (PEA) can measure CHST1/CHSY1 alongside hundreds of other proteins in minute tissue samples, enabling comprehensive protein network analysis. Nanobody technology offers smaller antibody formats with superior tissue penetration and reduced immunogenicity for in vivo imaging applications. Optogenetic antibody systems, where binding is controllable with light, allow temporal manipulation of CHST1/CHSY1 function in specific cellular compartments. Antibody-drug conjugates targeting CHSY1 in cancer cells represent a potential therapeutic avenue, particularly for colorectal cancers where CHSY1 is overexpressed. Tissue-cleared whole-organ imaging with fluorescently-labeled antibodies can map CHST1/CHSY1 distribution across entire organs while maintaining cellular resolution. Finally, antibody-based single-cell proteomics platforms enable correlation of CHST1/CHSY1 expression with dozens of other proteins at single-cell resolution, critical for understanding heterogeneity in cancer and immune cell populations .