GALNTL6 Antibody

What is GALNTL6 and what is its biological function?

GALNTL6, also known as GalNAc-T17 or GALNT17, is an enzyme that catalyzes the initial reaction in O-linked oligosaccharide biosynthesis. Specifically, it transfers an N-acetyl-D-galactosamine residue to serine or threonine residues on protein receptors . This post-translational modification is crucial for protein function and cellular processes. The GALNTL6 gene has gained attention in sports genomics research, with specific polymorphisms associated with athletic performance characteristics . Additionally, GALNTL6 has been implicated in cancer biology, particularly colorectal cancer, where its expression patterns differ from normal tissues .

What are the common characteristics of commercially available GALNTL6 antibodies?

Most commercially available GALNTL6 antibodies share several key characteristics. They are typically rabbit polyclonal antibodies that have undergone affinity purification . These antibodies are reactive against human GALNTL6 proteins, with many also cross-reacting with mouse and rat orthologs . Some antibodies demonstrate broader species reactivity, including cow, dog, horse, and even zebrafish . The applications commonly supported include Western blotting (WB), ELISA, and immunohistochemistry on paraffin-embedded tissues (IHC-P) . The immunogens used to generate these antibodies are typically recombinant protein fragments, such as amino acids 1-150 or 95-144 of the human GALNTL6 protein .

How is GALNTL6 protein expression detected in cells and tissues?

GALNTL6 protein expression can be detected through several methodologies. Western blotting is a common approach, with predicted band size typically around 70 kDa . When performing immunohistochemistry, GALNTL6 positive signals appear as "sand dots" predominantly localized in the cytoplasm of cells . Quantitative real-time PCR (qRT-PCR) can be used to assess GALNTL6 mRNA expression levels, with specific primers (Forward: 5'-GAC AAG ACA GTG GTG GTG AG; Reverse: 5' GAA GGT CAG GCT CCA GTC A) having been validated in previous studies . For immunohistochemistry evaluation, staining intensity is typically graded on a scale from 0 (no staining) to 3 (strong brown staining), with comprehensive scoring combining both staining intensity and the proportion of positive cells .

What are the optimal conditions for using GALNTL6 antibodies in Western blotting?

For Western blotting applications, GALNTL6 antibodies are typically used at dilutions ranging from 1:250 to 1:500 . The predicted molecular weight of the GALNTL6 protein is approximately 70 kDa, which should be considered when interpreting bands . When designing Western blotting experiments, researchers should include appropriate control lysates. Based on published research, validated positive controls include RT-4 cells, U-251 MG cells, human plasma, human liver lysate, and human tonsil lysate, all of which have demonstrated detectable GALNTL6 expression . To ensure optimal results, ECL (enhanced chemiluminescence) detection systems have been successfully applied for visualizing GALNTL6 protein bands . It's advisable to optimize the antibody concentration for each specific application and cell/tissue type being studied.

What controls should be incorporated when using GALNTL6 antibodies in research?

When designing experiments with GALNTL6 antibodies, several controls should be incorporated to ensure reliable results. For Western blotting, include both positive controls (cells or tissues known to express GALNTL6, such as human placenta, liver, or tonsil tissues) and negative controls (samples where the primary antibody is omitted) . For quantitative real-time PCR analysis, appropriate housekeeping genes should be used as internal controls - glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been validated for standard conditions, while β-actin is preferable for hypoxia and oxidative stress assays . For immunohistochemistry, adjacent normal mucosa (ANM) tissues serve as important comparison controls when studying GALNTL6 expression in cancer tissues . Additionally, peptide competition assays can be performed to confirm antibody specificity, where pre-incubating the antibody with its specific immunogen peptide should abolish specific staining.

How should immunohistochemistry with GALNTL6 antibodies be optimized and evaluated?

For immunohistochemistry applications, GALNTL6 antibodies have been successfully used at dilutions of approximately 1:500 on paraffin-embedded tissues . Staining evaluation should follow a systematic approach: first, assess the proportion of GALNTL6-positive cells by counting at least 1,000 cells in the region of interest . Then, grade the staining intensity according to standardized criteria: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown) . Calculate a composite score by multiplying the proportion score by the intensity score . For reliable results, evaluate multiple fields (at least 5 different areas) per tissue section and use the average score . Positive staining for GALNTL6 typically presents as cytoplasmic "sand dots" in cells, which serves as a characteristic pattern to confirm specific staining .

What is the association between GALNTL6 gene polymorphisms and athletic performance?

Research has revealed a significant association between the GALNTL6 gene rs558129 polymorphism and athletic performance characteristics. The T allele of this polymorphism is significantly overrepresented in power athletes (37.0%) compared to endurance athletes (29.3%; odds ratio [OR] = 1.4, p = 0.032) and controls (28.6%; OR = 1.5, p = 0.015) . This association is particularly pronounced in strength athletes, who show the highest frequency of the T allele (43.9%; OR = 1.9, p = 0.0067 compared with endurance athletes; OR = 2.0, p = 0.0036 compared with controls) . In contrast, the C allele of rs558129 has been associated with endurance performance in Olympic and world-level endurance athletes (OR = 1.23) . Functional studies using the Wingate Anaerobic Test (WAnT) have demonstrated that T allele carriers exhibit 5-7% higher absolute and relative mean power and peak power compared to individuals with the CC genotype . These findings suggest that the GALNTL6 gene may influence energy metabolism pathways relevant to different types of athletic performance.

How is GALNTL6 expression altered in colorectal cancer?

GALNTL6 expression shows significant alterations in colorectal cancer (CRC) compared to normal tissues. Studies have demonstrated higher GALNTL6 mRNA and protein expression in certain CRC cell lines, including LOVO, HT29, and SW620, while lower expression was observed in SW480 and SW1116 compared to normal human colonic epithelial cell line NCM460 . Analysis of fresh CRC tissues consistently revealed significantly higher GALNTL6 expression in all cancer samples compared to their respective adjacent normal mucosa (ANM) . Immunohistochemical studies found GALNTL6 positive signals in 69% (58 out of 84) of paraffin-embedded CRC tissues versus only 14% (11 out of 77) of ANM tissues . The expression of GALNTL6 also correlates with clinicopathological features of CRC, particularly showing significant associations with tumor size and histological differentiation . Specifically, smaller tumors (<5 cm) and well/moderately differentiated tumors exhibit higher GALNTL6 expression compared to larger or poorly differentiated tumors . This data points to a potential role of GALNTL6 in CRC development and progression.

What methodologies are effective for studying GALNTL6 gene polymorphisms?

Several methodologies have proven effective for studying GALNTL6 gene polymorphisms, particularly the rs558129 variant. Genome-wide association studies (GWAS) have been instrumental in initially identifying the association between GALNTL6 rs558129 and endurance performance . For targeted genotyping of this polymorphism in research or clinical settings, PCR-based methods followed by restriction fragment length polymorphism (RFLP) analysis or direct sequencing are commonly employed . Case-control study designs have proven effective for evaluating the association between GALNTL6 genotypes and specific phenotypes, such as athletic performance . When conducting such studies, it's essential to ensure that genotype distributions meet Hardy-Weinberg equilibrium expectations (p > 0.05) to validate the integrity of the data . For functional validation of genotype-phenotype associations, performance tests such as the Wingate Anaerobic Test (WAnT) provide quantitative measures of anaerobic power that can be correlated with specific genotypes . This multi-method approach combining genetic analysis with functional testing provides more robust evidence for the role of GALNTL6 variants in human performance.

What are common challenges in GALNTL6 antibody experiments and how can they be addressed?

Researchers may encounter several challenges when working with GALNTL6 antibodies. One common issue is variable staining intensity across different tissue types, which can be addressed by optimizing antibody concentration for each specific tissue . For Western blotting applications, non-specific bands may appear; this can be minimized by using longer blocking times and more stringent washing conditions . When working with paraffin-embedded tissues, inadequate antigen retrieval can result in false-negative results. This can be overcome by testing multiple antigen retrieval methods (heat-induced versus enzymatic) and optimizing the retrieval buffer composition and pH . Background staining in immunohistochemistry can be reduced by using appropriate blocking sera, optimizing antibody dilutions, and including 0.1-0.3% Triton X-100 in antibody diluents to improve penetration and reduce non-specific binding . For quantitative analysis, inconsistent housekeeping gene expression under certain conditions (like hypoxia) may confound results, so selection of appropriate internal controls is critical—β-actin has been validated for hypoxia and oxidative stress assays with GALNTL6 .

What are the key considerations for studying GALNTL6 in different experimental models?

When studying GALNTL6 across different experimental models, researchers should consider several key factors. Species-specific differences in GALNTL6 sequence and expression patterns exist, so antibody cross-reactivity should be confirmed when transitioning between species models . Different cell lines may express varying levels of GALNTL6—established baselines include higher expression in LOVO, HT29, and SW620 CRC lines and lower expression in SW480 and SW1116 lines compared to normal colonic epithelial cells . For in vivo models, tissue-specific expression patterns should be considered, as GALNTL6 shows differential expression across tissue types . When studying genetic associations, population stratification may influence results, so researchers should ensure appropriate matching of case and control populations . Environmental factors may modulate GALNTL6 expression or function, particularly in studies related to athletic performance where training status may confound genetic associations . Finally, when designing experiments to study GALNTL6 function, consider its biochemical role in O-linked glycosylation and select appropriate experimental readouts that reflect this enzymatic activity .

What are promising areas for future GALNTL6 research?

Several promising research directions could expand our understanding of GALNTL6 function and applications. The proposed relationship between GALNTL6 polymorphisms, gut microbiome, and short-chain fatty acid (SCFA) regulation merits further investigation, particularly regarding anti-inflammatory functions and lactate resynthesis in athletic performance . Exploring the mechanistic link between GALNTL6 expression and colorectal cancer progression could yield valuable insights into cancer biology and potential therapeutic targets . Studies investigating how GALNTL6-mediated O-linked glycosylation affects protein function, cellular signaling, and physiological processes would enhance our fundamental understanding of this enzyme's biological role . Development of more specific monoclonal antibodies targeting different GALNTL6 epitopes would improve detection specificity and enable more precise localization studies . Multi-omics approaches integrating genomics, proteomics, and metabolomics could provide a more comprehensive view of GALNTL6's role in health and disease . Finally, functional studies using gene editing technologies (CRISPR/Cas9) to modulate GALNTL6 expression or introduce specific polymorphisms would help establish causality in observed associations with athletic performance and disease states .