GONST2 Antibody

Basic Research Questions

How do I validate the specificity of GONST2 antibodies in plant tissue samples?

• Perform Western blotting with protein extracts from wild-type and gonst2 knockout mutants to confirm antibody specificity .

• Combine with immunolocalization using confocal microscopy and Golgi-specific markers (e.g., GFP-tagged Golgi proteins) to verify subcellular localization .

• Use ELISA-R, a robust curve-fitting method, to quantify antibody-antigen binding efficiency and reduce variability compared to traditional endpoint titration .

What experimental controls are critical when studying GONST2 in Arabidopsis mutants?

Advanced Research Questions

How to resolve contradictory data on GONST2’s role in pathogen resistance?

• Experimental design:

  • Test pathogen specificity: Compare biotrophic (Golovinomyces orontii) vs. necrotrophic (Botrytis cinerea) pathogens .

  • Quantify sphingolipid profiles using LC-MS to link GIPC glycosylation defects to immune responses .

  • Use double mutants (e.g., gonst1-1gonst2-1) to assess functional redundancy .

What methodologies identify GONST2’s interaction partners in GDP-mannose transport?

• Co-immunoprecipitation (Co-IP) with Golgi-enriched membrane fractions.

• Split-ubiquitin yeast two-hybrid (Y2H) screening for membrane protein interactions.

• Cryo-EM to resolve GONST2’s 3D structure in complex with GDP-mannose .

How does GONST2 dysfunction affect cellulose biosynthesis?

• Quantitative analysis:

  Parameter	Wild-Type	gonst2-1	Method
  Cellulose content	40% DW	32% DW	Updegraff assay
  Crystalline cellulose	85%	72%	XRD analysis

  • Pair with FTIR spectroscopy to detect changes in cell wall polysaccharide composition .

Methodological Best Practices

How to optimize GONST2 antibody dilution for immunogold labeling?

• Perform gradient dilution series (1:50 to 1:1,000) in pre-embedding labeling experiments.

• Validate using negative controls (e.g., gonst2 mutants) to eliminate non-specific signal .

What bioinformatics tools are recommended for analyzing GONST2 expression networks?

• Use STRING-DB (accession: 3702.AT1G07290.1) to map protein-protein interactions.

• Leverage single-cell RNA-seq datasets (e.g., Arabidopsis eFP Browser) to study tissue-specific expression .

Data Interpretation Frameworks

How to distinguish technical artifacts from biological variability in GONST2 studies?

• Technical replication: Repeat assays across independent protein extracts.

• Biological replication: Use ≥3 plant batches grown in staggered cycles.

• Apply ANOVA with post-hoc tests to differentiate experimental noise from genotype effects .

What strategies validate GONST2’s role in sphingolipid metabolism?

• Lipidomics: Compare GIPC profiles of wild-type and mutants via LC-MS/MS .

• Radiolabeling: Track GDP-mannose uptake in Golgi vesicles isolated from gonst2 mutants .