B3GALT4 Antibody

Here’s a structured collection of FAQs addressing academic research considerations for B3GALT4 antibodies, synthesized from scientific literature and technical specifications:

How do I validate B3GALT4 antibody specificity for Western blot (WB) experiments?

• Methodological approach:

  • Perform peptide competition assays using the immunogen sequence (e.g., residues 80-270 of human B3GALT4 ).

  • Validate using B3GALT4-overexpressing cell lysates (e.g., MDA-MB-468 or MCF-7 lines ) and compare with knockout controls.

  • Check for a single band at ~42 kDa (canonical molecular weight) .

What are the optimal applications for B3GALT4 antibodies in immunohistochemistry (IHC)?

• Key protocols:

  • Use 1:50–1:200 dilutions for formalin-fixed paraffin-embedded (FFPE) tissues .

  • Antigen retrieval with citrate buffer (pH 6.0) improves epitope accessibility .

  • Prioritize antibodies validated for IHC-Paraffin (e.g., Novus Biologicals NBP2-14341 ).

How to resolve contradictory findings about B3GALT4’s role in cancer progression?

• Context:

  • Pro-tumorigenic role: In breast cancer, B3GALT4 overexpression promotes proliferation via AKT/mTOR pathway suppression of autophagy .

  • Anti-tumorigenic role: In neuroblastoma, B3GALT4 inhibits lipid raft formation, enhancing CD8+ T-cell infiltration via CXCL9/10 chemokines .

Resolution strategy:

• Use isoform-specific antibodies to confirm target specificity (e.g., anti-B3GALT4 [EPR11650] ).

• Compare tissue-specific glycosylation patterns affecting antibody-epitope binding .

What controls are critical for B3GALT4 antibody-based autophagy studies?

• Essential controls:

  • Positive: Starved cells (induces LC3-II accumulation).

  • Negative: Bafilomycin A1-treated cells (blocks autophagosome-lysosome fusion ).

  • Technical: Include siRNA-mediated B3GALT4 knockdown to confirm pathway specificity .

How to address cross-reactivity in multi-species studies?

• Validation framework:

  • Test reactivity against orthologs: Mouse (NP_001335162), Rat (XP_006245146), using recombinant proteins .

  • Use antibodies with confirmed cross-species reactivity (e.g., Fine Biotech Co.’s antibody ).

What functional assays link B3GALT4 activity to ganglioside biosynthesis?

• Advanced methodology:

  • Lipid raft isolation: Use sucrose density centrifugation to assess GD2 expression changes post-B3GALT4 modulation .

  • Metabolic labeling: Incorporate radiolabeled UDP-galactose to track ganglioside synthesis in B3GALT4-KO cells .

Why does B3GALT4 antibody yield variable results in flow cytometry?

• Solutions:

  • Optimize fixation/permeabilization: Methanol fixation improves intracellular epitope detection .

  • Validate with overexpression systems (e.g., HEK293T transient transfection ).

How to confirm B3GALT4’s Golgi localization?

• Multiplex imaging:

  • Co-stain with Golgin-97 or GM130 markers.

  • Use antibodies validated for immunofluorescence (IF) (e.g., antibodies-online anti-B3GALT4 ).