B4GALNT2 Antibody

What is B4GALNT2 and what cellular functions does it perform?

B4GALNT2 (β-1,4-N-acetylgalactosamine transferase 2) is an enzyme that catalyzes the transfer of beta-1,4-linked GalNAc to the galactose residue of an alpha-2,3-sialylated chain. It plays a crucial role in the biosynthesis of the human Sd(a) antigen, which is found on more than 90% of Caucasian red blood cells, and the Cad antigen, which is serologically and biochemically related to the Sd(a) antigen . This glycosyltransferase is expressed on erythrocyte surfaces and in body secretions. In cellular contexts, B4GALNT2 has been identified as affecting growth traits in various species and participating in cancer-related cellular processes .

What is the molecular weight of B4GALNT2 protein and how is it detected in Western blot analysis?

The B4GALNT2 protein is typically observed at a molecular weight of approximately 65 kDa when detected by Western blot analysis . For optimal detection, researchers should use SDS-PAGE followed by western blot with the appropriate antibody dilution (typically 1:500-1:5000 range). Validation studies have successfully detected B4GALNT2 in Jurkat cells and COLO 320 cells . When running western blots, it's advisable to use appropriate controls and standardized loading protocols to ensure accurate detection of the protein.

What are the recommended applications and dilutions for B4GALNT2 antibodies in different experimental techniques?

B4GALNT2 antibodies have been validated for multiple applications with specific recommended dilutions:

For optimal results in IHC applications, paraffin-embedded tissue sections should be used with the antibody at a dilution of approximately 1:50, as validated in human colon cancer tissue samples . The antibody demonstrates specific staining patterns that can be visualized under both low (10x) and high (40x) magnification.

How should B4GALNT2 antibodies be stored and handled to maintain optimal activity?

B4GALNT2 antibodies should be stored at -20°C and should NOT be aliquoted to maintain stability . The typical formulation includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, which helps preserve antibody functionality. When working with the antibody, minimize freeze-thaw cycles and keep on ice during experimental procedures. Prior to use, centrifuge the antibody vial briefly to ensure collection of the entire volume. For long-term projects, it's advisable to check antibody performance periodically against positive controls to confirm that reactivity is maintained.

How is B4GALNT2 expression altered in breast cancer, and what experimental methods have validated these findings?

Analysis of The Cancer Genome Atlas (TCGA) database has revealed that B4GALNT2 is highly expressed in breast cancer tissues compared to adjacent normal tissues . This differential expression was confirmed through multiple approaches:

• Genetic analysis of 1094 breast cancer and normal tissue samples from the TCGA database

• Analysis of 106 pairs of matched breast cancer and adjacent normal tissues

• RT-qPCR assessment of B4GALNT2 mRNA expression in five different breast cancer cell lines

The overexpression was statistically significant (p<0.05) and was visualized through volcano plots of differential gene expression and paired expression diagrams . These findings suggest B4GALNT2 may serve as a potential biomarker for breast cancer detection or prognosis.

What functional effects have been observed following knockdown of B4GALNT2 in cancer cell lines?

Lentivirus-assisted knockdown of B4GALNT2 in triple negative breast cancer (TNBC) cell lines (HCC1937 and MDA-MB-231) has demonstrated significant functional effects:

• Inhibition of cell proliferation (approximately 2.0-fold in HCC1937 and 6.4-fold in MDA-MB-231 cells after 5 days)

• Decreased cell viability as measured by MTT assay

• Increased apoptosis rates detected by flow cytometry

• Reduced cell migration and invasion abilities as determined by Transwell assay

• Cell cycle arrest in G1 phase (increased G1 phase cells, decreased S phase cells, no change in G2/M phase)

The successful knockdown was confirmed by both RT-qPCR (showing ~70-80% reduction in mRNA levels) and Western blot analysis to verify decreased protein expression . These results collectively suggest that B4GALNT2 plays a pro-oncogenic role in breast cancer progression.

What protein interactions have been identified for B4GALNT2, and how do these interactions contribute to cellular function and disease mechanisms?

Co-immunoprecipitation (Co-IP) mass spectroscopy-assisted analysis has identified that the B4GALNT2 protein interacts with HLA-B protein, a product of the human leukocyte antigen (HLA) class I gene . This interaction appears to be functionally significant, as in vitro overexpression of HLA-B in B4GALNT2-knocked down MDA-MB-231 cell lines significantly recovered cell proliferation, viability, and migration abilities . This suggests that B4GALNT2 may promote breast cancer progression through its interaction with HLA-B protein in a downstream pathway mechanism. Further investigation of this interaction could provide insights into novel therapeutic approaches targeting this pathway.

How do genetic variants in B4GALNT2 affect its function and potential role in disease processes?

Genetic variants in B4GALNT2 can affect its function through multiple mechanisms. For example, a mutation in B4GALNT2 (G to A) resulted in:

• Altered mRNA secondary structure (visible changes in structural predictions)

• Increased minimum free energy from -2087.40 kJ/mol to -2078.20 kJ/mol

• Reduced mRNA stability after actinomycin D (ACTD) treatment, with significantly lower expression at 2h and 4h compared to wild type (p<0.05 or p<0.01)

These structural and stability changes can affect the expression and function of B4GALNT2, potentially altering its role in cellular processes. Additionally, B4GALNT2 expression has been found to change susceptibility to Salmonella infection and is considered a newly discovered antigen in xenotransplantation . Research methodologies for studying these variants typically involve site-directed mutagenesis, mRNA stability assays, and functional testing in relevant cell models.

What are the common challenges in optimizing immunohistochemistry protocols for B4GALNT2 antibodies, and how can they be addressed?

When performing immunohistochemistry with B4GALNT2 antibodies, researchers commonly encounter several challenges that can be addressed through methodical optimization:

• Background staining: Optimize blocking protocols using 5% serum from the same species as the secondary antibody. Include a peroxidase blocking step (3% H₂O₂ for 10 minutes) before primary antibody incubation.

• Antibody dilution: While the recommended dilution range is 1:20-1:200 for IHC , perform a dilution series experiment starting with 1:50 (as validated in colon cancer tissue) to determine optimal concentration for your specific tissue.

• Antigen retrieval: Test different antigen retrieval methods (heat-induced epitope retrieval at pH 6.0 vs. pH 9.0) as B4GALNT2 detection may vary in different tissues.

• Validation controls: Always include positive controls (human colon tissue has been validated ) and negative controls (primary antibody omission and isotype controls) to interpret results accurately.

• Signal amplification: For tissues with low B4GALNT2 expression, consider using polymer-HRP detection systems for signal amplification while maintaining specificity.

How should researchers interpret contradictory results between mRNA expression and protein detection of B4GALNT2 in experimental models?

When faced with discrepancies between B4GALNT2 mRNA and protein levels, consider the following methodological approach to resolve and interpret these contradictions:

• Temporal factors: The observed discrepancy may reflect time-dependent processes, as transcription precedes translation. Perform time-course experiments measuring both mRNA and protein levels.

• Post-transcriptional regulation: B4GALNT2 mRNA stability can be affected by mutations, as demonstrated by actinomycin D chase experiments showing differential decay rates between wild-type and mutant forms . Assess mRNA stability using actinomycin D treatment followed by RT-qPCR at different time points.

• Post-translational modifications: Investigate whether B4GALNT2 undergoes proteasomal degradation using proteasome inhibitors (e.g., MG132), which may reveal regulation at the protein level.

• Technical considerations:

  • Verify antibody specificity using knockdown/knockout controls, as demonstrated in lentivirus knockdown experiments

  • Confirm primer specificity for RT-qPCR

  • Use multiple detection methods (e.g., immunofluorescence in addition to Western blot)

• Experimental validation: Generate rescue experiments where B4GALNT2 is re-expressed in knockdown models to confirm specificity of observed phenotypes, similar to the HLA-B overexpression approach used in knocked-down MDA-MB-231 cells .

Understanding these multi-level regulatory mechanisms is essential for accurate interpretation of B4GALNT2 expression data in research contexts.