ST6GALNAC1 Antibody, Biotin conjugated

What is ST6GALNAC1 and what cellular functions does it regulate?

ST6GALNAC1 (ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1) is a 68.6 kDa glycosyltransferase enzyme localized primarily in the Golgi apparatus. The protein plays a crucial role in protein glycosylation by specifically conjugating sialic acid with an alpha-2-6 linkage to N-acetylgalactosamine (GalNAc) glycan chains attached to serine or threonine residues in glycoproteins . This sialyltransferase is particularly important in intestinal goblet cells where it catalyzes the formation of the sialyl-Tn (S-Tn) antigen . The enzyme's activity is essential for maintaining mucus integrity and protecting intestinal mucosa against excessive bacterial proteolytic degradation, thus playing a key role in intestinal host-commensal homeostasis . ST6GALNAC1 is widely expressed across multiple human tissues, with notable expression in the appendix and bronchus .

What are the key specifications of the biotin-conjugated ST6GALNAC1 antibody?

The biotin-conjugated ST6GALNAC1 antibody (catalog number ABIN1938467) is a polyclonal antibody generated in rabbits immunized with a KLH-conjugated synthetic peptide corresponding to amino acids 28-57 from the N-terminal region of human ST6GALNAC1 . This antibody demonstrates specific reactivity against human samples and has been validated for Western Blotting (WB) and ELISA applications . The antibody undergoes protein A purification to ensure high specificity and belongs to the IgG isotype . The biotin conjugation enhances detection sensitivity by allowing versatile secondary detection methods, including streptavidin-coupled fluorophores or enzymes. The specific binding region (amino acids 28-57) targets a highly conserved portion of the protein, making it suitable for detecting ST6GALNAC1 in various experimental contexts .

What optimization strategies are recommended for Western blot protocols using biotin-conjugated ST6GALNAC1 antibody?

When performing Western blot analysis with biotin-conjugated ST6GALNAC1 antibody, several critical optimization steps should be implemented. Begin with sample preparation by using RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors, as ST6GALNAC1 is subject to post-translational modifications . For optimal resolution of this 68.6 kDa protein, use 8-10% polyacrylamide gels and extend the separation time to achieve clear band discrimination from other glycosyltransferases with similar molecular weights . The transfer step should employ a wet transfer system at 30V overnight at 4°C to ensure complete transfer of higher molecular weight glycoproteins.

For blocking, use 5% BSA in TBST rather than milk, as milk contains glycoproteins that may interfere with glycosyltransferase detection . Dilute the biotin-conjugated ST6GALNAC1 antibody at 1:500 to 1:1000 in 1% BSA/TBST and incubate overnight at 4°C . Detection requires streptavidin-HRP (1:2000-1:5000) rather than traditional secondary antibodies. Include appropriate positive controls such as human colon or intestinal tissue lysates where ST6GALNAC1 is abundantly expressed . When interpreting results, verify the molecular weight against recombinant standards and remember that glycosylation states may cause slight shifts in apparent molecular weight.

How can the biotin-conjugated ST6GALNAC1 antibody be utilized in multi-parameter immunofluorescence studies?

For multi-parameter immunofluorescence studies, biotin-conjugated ST6GALNAC1 antibody offers exceptional versatility due to its compatibility with various streptavidin-conjugated fluorophores. Begin by fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 for intracellular Golgi localization . To overcome potential background issues, implement a streptavidin/biotin blocking kit before antibody incubation. The biotin-conjugated ST6GALNAC1 antibody should be used at 1:200-1:500 dilution and incubated overnight at 4°C .

For multi-parameter studies, combine with antibodies raised in species other than rabbit (such as mouse, goat, or rat) to avoid cross-reactivity. Use streptavidin conjugated to spectrally distinct fluorophores (Alexa Fluor 488, 555, or 647) for detection of the biotinylated antibody . For co-localization studies, pair with antibodies against Golgi markers (GM130), mucin proteins (MUC1, MUC2), or other glycosyltransferases (GALNTs) . When imaging, use sequential scanning to minimize spectral overlap, and include proper compensation controls. This approach allows simultaneous visualization of ST6GALNAC1 with interacting partners or subcellular compartments, enabling detailed analysis of protein trafficking and functional relationships in healthy versus diseased tissues.

What experimental approaches can validate miRNA regulation of ST6GALNAC1 expression identified through in silico analysis?

To experimentally validate the regulatory relationship between predicted miRNAs (miR-21-5p, miR-30e-5p, and miR-26b-5p) and ST6GALNAC1 expression identified through in silico analysis , a multi-faceted approach is required. Begin with luciferase reporter assays by cloning the 3'UTR of ST6GALNAC1 downstream of a luciferase reporter gene and co-transfecting with miRNA mimics or inhibitors. Site-directed mutagenesis of predicted miRNA binding sites can confirm direct interaction specificity.

For cellular validation, conduct gain- and loss-of-function experiments by transfecting colorectal cancer cell lines with miRNA mimics, inhibitors, or siRNAs targeting ST6GALNAC1. Measure changes in ST6GALNAC1 expression at both mRNA level via qRT-PCR and protein level using the biotin-conjugated ST6GALNAC1 antibody in Western blot or ELISA formats . To establish correlation in clinical samples, perform matched analysis of miRNA levels and ST6GALNAC1 protein expression in paired normal and colorectal cancer tissues using qRT-PCR for miRNAs and immunohistochemistry with the ST6GALNAC1 antibody .

For functional consequences, assess altered O-glycosylation patterns using lectins specific for sialyl-Tn antigen following miRNA manipulation. Additionally, investigate phenotypic changes in cell migration, invasion, and mucin production. This comprehensive approach will validate in silico predictions and establish the functional significance of miRNA-mediated regulation of ST6GALNAC1 in colorectal cancer progression.

How can researchers address cross-reactivity concerns with ST6GALNAC1 antibody in tissues expressing multiple sialyltransferases?

Cross-reactivity is a significant concern when studying sialyltransferases due to their structural similarities and conserved catalytic domains. When using ST6GALNAC1 antibody in tissues expressing multiple sialyltransferases, implement a multi-step validation approach. First, perform comprehensive blocking experiments by pre-incubating the antibody with recombinant ST6GALNAC1 protein (amino acids 28-57) to confirm binding specificity . This peptide competition assay should eliminate specific staining if the antibody is truly specific.

Additionally, validate specificity through knockdown/knockout controls by using siRNA or CRISPR-Cas9 to create ST6GALNAC1-deficient cell lines, confirming signal reduction with the antibody . When analyzing tissues, compare expression patterns with known ST6GALNAC1 distribution from RNA-seq databases and previously published findings . The antibody should demonstrate strongest reactivity in tissues like colon, gastric mucosa, and salivary glands where ST6GALNAC1 is highly expressed.

For precise discrimination from ST6GALNAC2, which shares sequence homology, combine the antibody detection with isoform-specific enzymatic activity assays that distinguish alpha-2,6-sialyltransferase activity from other sialyltransferases . When interpreting data, always cross-reference with complementary detection methods such as in situ hybridization for ST6GALNAC1 mRNA to confirm protein localization patterns match transcript distribution in tissues with complex glycosyltransferase expression profiles.

What factors affect the reproducibility of ST6GALNAC1 detection in colorectal cancer specimens, and how can they be controlled?

Reproducible detection of ST6GALNAC1 in colorectal cancer specimens faces multiple challenges that must be systematically addressed. Sample quality and preservation methods significantly impact detection reliability, as glycoproteins are sensitive to degradation. Standardize tissue collection using immediate fixation in 10% neutral buffered formalin for exactly 24 hours, followed by paraffin embedding to preserve antigenic properties . For frozen specimens, snap freezing in liquid nitrogen and storage at -80°C with minimal freeze-thaw cycles is crucial.

Antigen retrieval methods must be optimized specifically for ST6GALNAC1 detection. Compare citrate buffer (pH 6.0) versus EDTA buffer (pH 9.0) heat-induced epitope retrieval to determine optimal conditions for exposing the N-terminal epitope (amino acids 28-57) . Implement rigorous antibody validation by including known positive controls (normal colon mucosa) and negative controls (ST6GALNAC1-negative tissues or isotype controls) in each experimental batch .

Tumor heterogeneity presents a significant challenge for reproducible detection. Implement tissue microarrays with multiple cores per tumor and establish quantitative scoring systems for immunohistochemistry that account for both staining intensity and percentage of positive cells . Inter-observer variability should be minimized through blinded assessment by multiple pathologists or automated image analysis software. Finally, correlate protein detection with transcript levels through parallel qRT-PCR analysis of ST6GALNAC1 mRNA from adjacent tissue sections to validate expression patterns . This comprehensive approach enhances reproducibility of ST6GALNAC1 detection across diverse colorectal cancer specimens and research settings.

How should researchers interpret discrepancies between ST6GALNAC1 expression levels and sialyl-Tn antigen presence in cancer studies?

The paradoxical observation that sialyl-Tn (STn) antigen levels do not always correlate with ST6GALNAC1 expression in cancer tissues requires careful interpretation within a broader context of glycobiology . When encountering such discrepancies, first verify the technical accuracy of both measurements by employing multiple detection methods. For ST6GALNAC1, combine the biotin-conjugated antibody detection with mRNA quantification using qRT-PCR . For STn antigen, use multiple anti-STn antibodies (clones HB-STn1, TKH2) alongside plant lectins like Maackia amurensis lectin that bind sialylated structures.

Consider post-transcriptional and post-translational regulatory mechanisms that may affect the relationship between gene expression and enzymatic activity. MicroRNA regulation, particularly by miR-21-5p, miR-30e-5p, and miR-26b-5p, may suppress translation without affecting mRNA levels . Additionally, protein stability, trafficking to the Golgi apparatus, and post-translational modifications can impact enzyme activity without changing expression levels.

Evaluate the substrate availability landscape, as STn synthesis requires both ST6GALNAC1 and appropriate O-glycan precursor structures generated by GALNTs . Altered expression of competing glycosyltransferases that use the same substrates may redirect the glycosylation pathway. Analyze the expression patterns of ST6GALNAC2 and other sialyltransferases that might compensate for ST6GALNAC1 downregulation .

Finally, consider the role of the tumor microenvironment, as pH changes, hypoxia, and inflammatory signals can alter glycosyltransferase activity independently of expression levels. These comprehensive considerations will help researchers interpret complex relationships between ST6GALNAC1 expression and STn antigen presence in cancer studies, potentially revealing novel regulatory mechanisms of cancer-associated glycosylation.

What emerging applications of ST6GALNAC1 antibodies could advance glycosylation-targeted cancer therapies?

The biotin-conjugated ST6GALNAC1 antibody offers promising applications for developing novel glycosylation-targeted cancer therapies. Researchers can utilize this antibody for high-throughput screening of small molecule inhibitors that modulate ST6GALNAC1 activity without affecting other sialyltransferases . By immobilizing the antibody on biosensor chips, surface plasmon resonance (SPR) can identify compounds that alter enzyme conformation or substrate binding, potentially disrupting aberrant glycosylation in cancer cells.

The antibody also enables precise patient stratification for glycosylation-targeted therapies through immunohistochemical analysis of tumor biopsies . This approach can identify patients with altered ST6GALNAC1 expression patterns who might benefit from specific glycosylation-modulating treatments. For developing antibody-drug conjugates (ADCs), the biotin-conjugated format provides a platform for attaching cytotoxic payloads that target cells expressing surface STn antigens generated by ST6GALNAC1 .

In adoptive cell therapy approaches, the antibody can help characterize engineered T cells expressing chimeric antigen receptors (CARs) that recognize STn antigens on cancer cells. Additionally, researchers can employ the antibody to develop "glyco-vaccines" by coupling STn-carrying peptides with immune adjuvants, potentially training the immune system to recognize and eliminate cancer cells expressing these altered glycans . These emerging applications highlight how ST6GALNAC1 antibodies could bridge fundamental glycobiology research with translational cancer therapeutic development targeting aberrant glycosylation signatures.

How might single-cell glycomics benefit from ST6GALNAC1 antibody-based detection systems?

Single-cell glycomics represents a frontier in glycobiology research, and ST6GALNAC1 antibody-based detection systems could dramatically enhance this emerging field. The biotin-conjugated format is particularly advantageous for developing microfluidic antibody capture assays that can isolate and characterize individual cells based on their ST6GALNAC1 expression levels . This approach would enable selection of specific cell populations with distinct sialylation patterns for subsequent glycomic analysis.

When integrated with cyclic immunofluorescence (CycIF) technologies, the antibody allows multiplexed detection of ST6GALNAC1 alongside other glycosyltransferases and glycan epitopes within the same tissue section or cell preparation . This enables spatial mapping of glycosylation enzyme networks at single-cell resolution. For mass cytometry (CyTOF) applications, the biotin-conjugated antibody can be labeled with metal isotopes, allowing simultaneous quantification of ST6GALNAC1 with dozens of other cellular markers to correlate enzyme expression with specific cellular phenotypes and functional states .

Coupling the antibody with proximity ligation assays (PLA) would reveal protein-protein interactions between ST6GALNAC1 and other glycosylation machinery components within individual cells, illuminating the spatial organization of the O-glycosylation apparatus . These advanced applications would provide unprecedented insights into cellular heterogeneity of glycosylation processes in both normal physiology and disease states, potentially revealing new subpopulations of cells with distinct glycosylation signatures that could serve as targets for precision medicine approaches.

What key considerations should researchers prioritize when selecting and implementing ST6GALNAC1 antibody-based methodologies?

When selecting and implementing ST6GALNAC1 antibody-based methodologies, researchers should prioritize several critical considerations to ensure experimental success and data reliability. First, match the antibody specificity to your specific research question - the biotin-conjugated antibody targeting amino acids 28-57 of the N-terminal region provides excellent specificity for human ST6GALNAC1, but may have limited cross-reactivity with orthologous proteins in other species . For evolutionary studies or animal models, antibodies targeting more conserved regions might be preferable.

Consider the detection method compatibility based on your experimental platform. The biotin conjugation offers versatility with streptavidin-based detection systems, but may not be ideal for all applications . For multiplexed immunofluorescence where multiple biotin-labeled antibodies might be used, alternative conjugates or unconjugated primary antibodies might prevent signal overlap. Validate the antibody rigorously in your specific experimental system using positive and negative controls, including ST6GALNAC1 knockdown/knockout samples .