GBF1 Antibody

Basic Research Questions

How to validate GBF1 antibody specificity in Western blot for different cellular models?

• Methodological approach:

  • Perform siRNA-mediated GBF1 knockdown alongside non-targeting controls. Compare band intensity reduction at ~200 kDa (GBF1's molecular weight) .

  • Use overexpression constructs (e.g., YFP-GBF1 ) to confirm antibody recognition of tagged vs. endogenous protein.

  • Test cross-reactivity with lysates from GBF1-knockout cell lines or tissues.

What experimental controls are critical for GBF1 immunoprecipitation (IP) assays?

• Essential controls:

  • IgG isotype control to rule out nonspecific binding.

  • Input lysate (10% of IP volume) to confirm target presence.

  • Post-IP supernatant to verify depletion efficiency.

  • Co-IP validation with known interactors (e.g., C/EBP-β ).

How to optimize GBF1 antibody dilution for immunofluorescence (IF)?

• Protocol refinement:

  • Test a dilution series (1:100–1:1000) in fixed HeLa cells, monitoring Golgi-localized signal (GBF1's primary site ).

  • Compare with BFA-treated cells: GBF1 should accumulate on Golgi membranes upon BFA exposure .

  • Validate using cells expressing YFP-GBF1 to confirm colocalization .

Advanced Research Questions

How to resolve contradictions in GBF1 localization data across infection models?

• Analytical framework:

  Condition	Localization Pattern	Key Variables
  Poliovirus infection	Redistribution to replication organelles	Viral 3A protein interaction
  Steady-state	Golgi apparatus	Cell type, secretory activity
  BFA treatment	Stabilized Golgi association	BFA concentration, exposure time

  • Use compartment-specific markers (e.g., GM130 for Golgi) and super-resolution microscopy.

What strategies confirm GBF1's role in Arf GTPase activation pathways?

• Functional assays:

  • Arf-GTP pulldown: Compare Arf activation in GBF1-depleted vs. wild-type cells using GST-GGA3 or MitoPB1 domains .

  • Dominant-negative mutants: Express GBF1-E794K (catalytically inactive) to block Arf activation .

  • BFA resistance assays: Monitor viral replication efficiency in GBF1-overexpressing cells .

How to investigate GBF1 phosphorylation dynamics in disease contexts?

• Phosphoproteomic workflow:

  • Generate phosphorylation-deficient mutants (e.g., Ser/Thr→Ala) based on mass spectrometry data .

  • Use Phos-tag™ SDS-PAGE to separate phosphorylated isoforms.

  • Pair with kinase inhibitors (e.g., CDK1/5 inhibitors) to map regulatory pathways.

Methodological Considerations for Data Interpretation

How to differentiate artifact vs. true signal in GBF1 antibody-based assays?

• Validation matrix:

  Artifact Source	Detection Method	Mitigation Strategy
  Cross-reactive epitopes	Knockout validation	Use multiple antibodies targeting different domains
  Protein aggregation	Solubility fractionation	Include 1% Triton X-100 in lysis buffer
  Post-translational modification interference	Phosphatase treatment	Compare signal with/without λ-phosphatase

What approaches validate GBF1's interaction with viral proteins like poliovirus 3A?

• Multi-modal verification:

  • Proximity ligation assay (PLA): Quantify GBF1-3A interaction foci in infected cells .

  • Structural mapping: Co-express truncated 3A mutants (e.g., ΔN-terminal) with GBF1 to identify binding domains .

  • Functional rescue: Test if GBF1 mutants (e.g., A795E ) restore viral replication in GBF1-depleted cells.

Key Citations for Experimental Design

• Golgi dynamics and BFA resistance:

• Viral interface studies:

• Antibody validation protocols: