FLOT1 Antibody

Here’s a structured collection of FAQs for FLOT1 antibody research, synthesized from peer-reviewed studies and technical documentation:

How to validate FLOT1 antibody specificity in Western blotting (WB)?

• Method:

  • Run a knockout (KO) control lysate alongside experimental samples.

  • Compare bands in wild-type vs. FLOT1-KO cell lines (e.g., HCC cell lines ).

  • Confirm the observed molecular weight (~47 kDa) matches expected size .

• Troubleshooting:

  • Non-specific bands: Optimize blocking buffer (5% BSA vs. non-fat milk) and adjust antibody dilution (1:2,000–1:10,000 ).

  • Weak signal: Use fresh PFA fixation for tissue samples to preserve epitopes .

What antigen retrieval methods optimize FLOT1 detection in immunohistochemistry (IHC)?

• Protocol:

  Buffer	pH	Tissue Type	Citation
  TE buffer	9.0	Human stomach
  Citrate buffer	6.0	Paraffin-embedded sections

• Heat-induced epitope retrieval (HIER) is critical for formalin-fixed tissues. Validate with positive controls (e.g., human stomach tissue ).

How to resolve discrepancies in FLOT1 expression across cancer models?

• Case Study:

  • In hepatocellular carcinoma (HCC), FLOT1 is upregulated in metastatic cell lines (MHCC97H, HCCLM6) vs. normal liver cells (Lo2) .

  • Strategy:

    • Normalize to housekeeping genes (e.g., GAPDH) across replicates.

    • Use dual validation (WB + RT-qPCR) to confirm transcriptional vs. post-translational regulation .

Can FLOT1 antibodies cross-react with non-target species in translational models?

• Data:

  Antibody	Validated Species	Unvalidated but Tested	Outcome
  PA2033	Human, Mouse, Rat	Dog (customer-reported)	Inconclusive; requires KO validation
  15571-1-AP	Human, Mouse, Rat	N/A	No cross-reactivity data beyond cited species

• Recommendation: Perform sequence alignment of FLOT1 epitopes between species before testing.

How to design a FLOT1/FLOT2 complex study in autoimmune diseases?

• Approach:

  • Co-transfect HEK293 cells with FLOT1/FLOT2 plasmids .

  • Use dual immunofluorescence with patient sera (1:10 dilution) and commercial antibodies (e.g., Sigma FLOT1/FLOT2) .

  • Quantify co-localization using confocal microscopy and software (e.g., ImageJ).

What controls are essential for FLOT1 functional assays in cancer progression?

• Key Controls:

  • Positive: High-FLOT1-expressing HCC cell lines (e.g., MHCC97H ).

  • Negative: FLOT1-silenced cells (siRNA/CRISPR).

  • Clinical correlation: Compare FLOT1 levels with patient survival data (low FLOT1 = longer survival ).

How to optimize immunoprecipitation (IP) for FLOT1 interactome studies?

• Protocol:

  • Lysate preparation: Use mild detergents (1% NP-40) to preserve protein complexes.

  • Antibody ratio: 0.5–4.0 µg antibody per 1–3 mg lysate .

  • Validate interactions via Co-IP followed by mass spectrometry.

Addressing inconsistent FLOT1 localization reports in membrane vs. cytoplasmic compartments

• Factors to Consider:

  • Fixation method: Prolonged formalin fixation may alter membrane protein localization .

  • Cell confluency: FLOT1 redistributes during cell polarization (validate with subcellular fractionation + WB).