ST6GALNAC6 Antibody

What is ST6GALNAC6 and what cellular functions does it perform?

ST6GALNAC6 (ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 6) is a glycosyltransferase enzyme that transfers sialyl groups from CMP-NeuAc onto glycoproteins and glycolipids, forming an alpha-2,6-linkage. This enzyme plays a significant role in the metabolism of lipids and protein glycosylation. It functions by producing branched type disialyl structures through transferring a sialyl group onto GalNAc or GlcNAc residues inside backbone core chains with terminal alpha-2,3-linked sialic acid on galactose. ST6GALNAC6 predominantly catalyzes the biosynthesis of ganglioside GD1alpha from GM1b and shows preference for glycolipids over glycoproteins .

Where is ST6GALNAC6 expressed and what is its subcellular localization?

ST6GALNAC6 is primarily expressed in the kidney, specifically in proximal tubule epithelial cells, as well as in colon cell lines. The protein has a wide tissue distribution and is reportedly expressed in various tissues including brain, heart, liver, lung, muscles, placenta, intestine, spleen, stomach, and testis. At the subcellular level, ST6GALNAC6 is localized to the Golgi apparatus membrane, where it functions as a single-pass type II membrane protein .

What are the key characteristics of ST6GALNAC6 protein structure?

The canonical ST6GALNAC6 protein in humans has 333 amino acid residues with a molecular mass of approximately 38.1 kDa. The protein contains several functional domains and may undergo post-translational modifications, particularly glycosylation. Up to three different isoforms have been reported for this protein. ST6GALNAC6 is a member of the Glycosyltransferase 29 protein family. The protein contains sequence motifs characteristic of sialyltransferases and has conserved cysteine residues important for maintaining its tertiary structure .

What are the primary research applications for ST6GALNAC6 antibodies?

ST6GALNAC6 antibodies are primarily used for immunodetection of the ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 6 protein in various research applications. The most common application is Western Blot (WB), which allows for the detection and semi-quantification of the protein in cell or tissue lysates. Other significant applications include Enzyme-Linked Immunosorbent Assay (ELISA) and Immunohistochemistry (IHC), which enable the detection of ST6GALNAC6 in solution or in tissue sections, respectively. The antibodies can also be used in immunofluorescence studies to determine the subcellular localization of the protein .

What is the optimal protocol for Western Blot analysis using ST6GALNAC6 antibodies?

For optimal Western Blot results with ST6GALNAC6 antibodies, researchers should follow these methodological steps:

• Prepare cell/tissue lysates under reducing conditions

• Separate proteins using SDS-PAGE (expect ST6GALNAC6 to migrate at approximately 36-38 kDa)

• Transfer proteins to a PVDF membrane

• Block the membrane with a suitable blocking buffer (typically containing 2% sucrose)

• Incubate with primary ST6GALNAC6 antibody at a concentration of 1-2 µg/mL

• Wash thoroughly to remove unbound antibody

• Incubate with an appropriate HRP-conjugated secondary antibody

• Develop using an Immunoblot Buffer Group 1 or equivalent detection system

This protocol has been demonstrated to detect a specific band for ST6GALNAC6 at approximately 36-38 kDa in various cell lines, including MDA-MB-231 human breast cancer cells .

How should ST6GALNAC6 antibodies be stored and handled to maintain optimal activity?

For maximum stability and activity, ST6GALNAC6 antibodies should typically be stored at -20°C. Liquid formulations are often supplied in 1x PBS buffer with 0.09% (w/v) sodium azide and 2% sucrose. For reconstitution of lyophilized antibodies, add the recommended volume (typically 50 µl) of distilled water to achieve a final concentration of 1 mg/ml in PBS buffer with 2% sucrose. To avoid loss of activity, minimize freeze-thaw cycles. For short-term storage (less than 1 week), antibodies may be kept at 4°C, but long-term storage should be at -20°C. Before use, allow the antibody to equilibrate to room temperature and centrifuge briefly if necessary to collect the solution at the bottom of the vial .

What are the common cross-reactivity issues with ST6GALNAC6 antibodies and how can they be addressed?

ST6GALNAC6 antibodies may cross-react with other members of the sialyltransferase family, particularly those with high sequence homology like ST6GALNAC1-5. Cross-reactivity can be addressed through several approaches:

• Select antibodies raised against unique epitopes in the N-terminal or C-terminal regions of ST6GALNAC6 that have low homology with other family members

• Validate antibody specificity using positive and negative controls (e.g., tissues/cells known to express or not express ST6GALNAC6)

• Perform pre-absorption tests with recombinant ST6GALNAC6 protein

• Use knockout/knockdown cell lines to confirm antibody specificity

• Compare reactivity patterns with antibodies from different manufacturers or clones

Additionally, researchers should be aware that different antibodies may show species-specific reactivity patterns. For instance, some ST6GALNAC6 antibodies react with human, mouse, rat, rabbit, horse, bovine, guinea pig, and dog samples, while others might have more limited cross-species reactivity .

What factors affect the reproducibility of immunodetection experiments using ST6GALNAC6 antibodies?

Several factors can impact the reproducibility of experiments using ST6GALNAC6 antibodies:

• Sample preparation methods: Different lysis buffers or protein extraction protocols may affect protein conformation and epitope accessibility

• Protein denaturation conditions: Over-heating samples may cause protein aggregation

• Blocking reagents: Inappropriate blocking can lead to high background or reduced specific signal

• Antibody concentration: Optimal dilutions should be determined empirically for each application

• Incubation conditions: Temperature, duration, and buffer composition can affect antibody binding

• Detection systems: Different secondary antibodies or visualization methods may vary in sensitivity

• Post-translational modifications: Glycosylation of ST6GALNAC6 may mask epitopes

• Batch-to-batch variations: Especially relevant for polyclonal antibodies

To enhance reproducibility, researchers should carefully optimize each step of their protocol and maintain consistent conditions across experiments. Detailed record-keeping of reagent sources, lot numbers, and experimental conditions is essential .

How can you distinguish between ST6GALNAC6 and other ST6GALNAC family members in immunodetection experiments?

Distinguishing between ST6GALNAC6 and other family members requires careful experimental design:

• Select highly specific antibodies targeting unique regions of ST6GALNAC6 not conserved in other family members

• Use control samples expressing only specific ST6GALNAC family members

• Employ molecular weight analysis (ST6GALNAC6 is approximately 38.1 kDa, which may differ from other family members)

• Perform parallel experiments with antibodies specific to different ST6GALNAC family members

• Validate results using complementary techniques such as mass spectrometry

• Consider tissue-specific expression patterns (ST6GALNAC6 is primarily expressed in kidney proximal tubule epithelial cells)

• Use RNA interference to specifically knock down ST6GALNAC6 and confirm antibody specificity

Additionally, researchers can leverage the subcellular localization patterns, as different family members may have subtle differences in their Golgi compartmentalization .

How can ST6GALNAC6 antibodies be utilized to investigate the role of sialylation in cancer biology?

ST6GALNAC6 antibodies can be powerful tools for investigating sialylation in cancer through several advanced approaches:

• Immunohistochemical analysis of cancer tissues to assess ST6GALNAC6 expression levels and correlate with clinical outcomes

• Co-localization studies with cancer-associated glycan structures to understand the relationship between ST6GALNAC6 expression and altered glycosylation

• Investigation of ST6GALNAC6's role in generating disialyl Lewis a, which has been suggested to be a cancer-associated antigen

• Analysis of ST6GALNAC6 expression in various cancer cell lines and comparison with normal counterparts

• Correlation studies between ST6GALNAC6 expression and cancer cell invasion or metastatic potential

• Development of tissue microarrays to screen ST6GALNAC6 expression across multiple cancer types

• Combined use with other markers to develop diagnostic or prognostic panels

These approaches can provide insights into how altered sialylation contributes to cancer progression and metastasis, potentially identifying new therapeutic targets or biomarkers .

What are the experimental considerations when studying ST6GALNAC6 interacting partners using immunoprecipitation?

When studying protein interactions with ST6GALNAC6 through immunoprecipitation, researchers should consider:

• Cell lysis conditions: Use mild detergents that preserve protein-protein interactions while effectively solubilizing membrane proteins from the Golgi

• Crosslinking options: Consider reversible crosslinking to capture transient interactions

• Control experiments: Include isotype controls and ST6GALNAC6-negative samples

• Pre-clearing steps: Reduce non-specific binding by pre-clearing lysates

• Antibody selection: Choose antibodies that do not interfere with protein interaction sites

• Washing stringency: Balance between removing non-specific interactions while preserving specific ones

• Elution methods: Consider native elution methods for downstream functional assays

• Detection strategies: Use mass spectrometry for unbiased identification of interacting partners

• Validation approaches: Confirm interactions through reciprocal immunoprecipitation or proximity ligation assays

Additionally, researchers should consider that ST6GALNAC6's transmembrane domain and Golgi localization may complicate the isolation of intact protein complexes, potentially requiring specialized approaches for membrane protein immunoprecipitation .

How can glycosyltransferase activity assays be combined with ST6GALNAC6 immunodetection to correlate protein levels with enzymatic function?

Integrating enzymatic activity assays with immunodetection of ST6GALNAC6 requires a multi-faceted approach:

• Sample preparation:

  • Prepare parallel samples from the same biological source

  • Carefully control lysis conditions to preserve enzyme activity for functional assays

• Activity measurement:

  • Utilize phosphatase-coupled methods to measure sialyltransferase activity

  • Use appropriate substrates such as fetuin from fetal calf serum or specific glycolipids

  • Measure the formation of disialylated structures from monosialylated precursors

• Protein quantification:

  • Perform quantitative Western blot analysis using calibrated standards

  • Use ELISA to precisely measure ST6GALNAC6 concentration in samples

• Correlation analysis:

  • Plot enzyme activity against protein levels to establish relationship

  • Account for potential post-translational modifications affecting activity

  • Consider the presence of endogenous inhibitors or activators

• Validation approaches:

  • Use recombinant ST6GALNAC6 at known concentrations as standards

  • Employ ST6GALNAC6 knockout/knockdown models as negative controls

  • Include specific inhibitors to confirm activity specificity

This integrated approach allows researchers to determine whether changes in ST6GALNAC6 activity correlate directly with protein levels or are affected by other regulatory mechanisms .

How can multi-omics approaches incorporate ST6GALNAC6 antibody-based research to understand sialylation networks?

Integrating ST6GALNAC6 antibody-based research into multi-omics frameworks provides powerful insights into sialylation networks:

• Glycoproteomics integration:

  • Use ST6GALNAC6 immunoprecipitation followed by mass spectrometry to identify specifically sialylated substrates

  • Correlate ST6GALNAC6 expression with changes in the sialylated glycoproteome

• Transcriptomics correlation:

  • Compare ST6GALNAC6 protein levels detected by antibodies with mRNA expression

  • Identify potential transcriptional regulatory networks governing ST6GALNAC6 expression

• Metabolomics connections:

  • Link ST6GALNAC6 protein levels with changes in sialic acid metabolism

  • Track the flow of metabolic precursors through the sialylation pathway

• Systems biology modeling:

  • Incorporate antibody-derived quantitative data on ST6GALNAC6 into mathematical models

  • Predict the impact of ST6GALNAC6 expression changes on broader glycosylation networks

• Spatial glycomics:

  • Combine ST6GALNAC6 immunohistochemistry with glycan imaging techniques

  • Map the spatial distribution of enzyme expression relative to its glycan products

This multi-faceted approach provides a comprehensive understanding of how ST6GALNAC6 functions within the broader context of cellular glycosylation processes and signaling networks .

What methodological approaches can be used to investigate the relationship between ST6GALNAC6 and other glycosyltransferases in sequential glycosylation processes?

Investigating ST6GALNAC6's relationship with other glycosyltransferases requires sophisticated methodological approaches:

• Co-localization studies:

  • Use dual immunofluorescence with antibodies against ST6GALNAC6 and other glycosyltransferases

  • Apply super-resolution microscopy to precisely map relative positions within the Golgi apparatus

• Enzymatic competition assays:

  • Measure ST6GALNAC6 activity in the presence of varying amounts of other glycosyltransferases

  • Assess substrate competition between sequential enzymes

• Proximity-based protein interaction studies:

  • Apply BioID or APEX2 proximity labeling to identify glycosyltransferases physically close to ST6GALNAC6

  • Use Förster Resonance Energy Transfer (FRET) to detect direct interactions

• Sequential knockdown/overexpression experiments:

  • Modulate expression of upstream or downstream glycosyltransferases and measure impact on ST6GALNAC6 function

  • Create double knockout models to assess compensatory mechanisms

• Glycan structural analysis:

  • Compare glycan profiles after selective inhibition or activation of specific glycosyltransferases

  • Use mass spectrometry to track the sequential addition of glycan residues

These approaches collectively provide insights into how ST6GALNAC6 coordinates with other glycosyltransferases in the complex process of glycan assembly within the Golgi apparatus .

What are the methodological considerations when using ST6GALNAC6 antibodies in high-throughput screening approaches for drug discovery?

Using ST6GALNAC6 antibodies in high-throughput screening for drug discovery requires careful methodological planning:

• Assay development:

  • Optimize antibody concentration and detection systems for maximum signal-to-noise ratio

  • Develop robust ELISA or automated Western blot protocols suitable for high-throughput formats

  • Consider cell-based immunofluorescence assays for compound screening

• Screening strategy considerations:

  • Select appropriate positive and negative controls for each plate

  • Implement Z-factor analysis to ensure assay quality

  • Include dose-response curves for promising compounds

• Technology platform selection:

  • Choose between cell-based vs. biochemical assays based on research goals

  • Consider multiplexed approaches to simultaneously measure ST6GALNAC6 protein levels and activity

  • Develop automated image analysis algorithms for high-content screening approaches

• Hit validation methodology:

  • Confirm hits using orthogonal antibodies targeting different epitopes

  • Evaluate effects on ST6GALNAC6 expression vs. enzymatic activity

  • Assess specificity by testing effects on related sialyltransferases

• Data analysis frameworks:

  • Implement machine learning algorithms to identify structure-activity relationships

  • Develop bioinformatic pipelines to integrate results with existing glycobiology databases

  • Create visualization tools specific to glycosyltransferase network perturbations