SUFE1 Antibody

Below is a curated collection of FAQs addressing key aspects of SUFU antibody research, organized by complexity and methodological focus. The responses integrate experimental design principles, data interpretation strategies, and technical considerations from peer-reviewed studies.

How to validate SUFU antibody specificity in Western blot experiments?

• Methodology:

  • Use knockout controls (e.g., SUFU KO HAP1 or HEK293T cells ) alongside wild-type lysates.

  • Include a loading control (e.g., GAPDH) to normalize protein levels.

  • Verify predicted band size (53 kDa) against observed molecular weight.

  • Example data:

    Sample Type	Band Observed (kDa)	Signal Intensity (vs Wildtype)
    Wildtype HAP1	53	100%
    SUFU KO HAP1	Absent	<5%
    Wildtype HEK293T	53	95%

  • Critical step: Compare with isotype controls (e.g., Rabbit IgG [EPR25A]) to rule out nonspecific binding .

What are the optimal conditions for intracellular SUFU detection via flow cytometry?

• Protocol optimization:

  • Fixation: 4% paraformaldehyde.

  • Permeabilization: 90% methanol.

  • Antibody dilution: 1:500 (0.1 µg/mL) .

  • Controls:

    • Unlabelled cells (autofluorescence baseline).

    • Isotype-matched antibodies (non-specific binding baseline).

How to resolve contradictory SUFU localization data between Western blot and immunofluorescence?

• Troubleshooting framework:

  • Cross-validate antibodies: Use orthogonal methods (e.g., CRISPR-Cas9 KO validation ).

  • Assess post-translational modifications: SUFU may undergo phosphorylation or ubiquitination, altering migration patterns.

  • Buffer compatibility: Ensure lysis buffers (e.g., RIPA) preserve epitope integrity.

Can SUFU antibodies distinguish between isoforms or post-translationally modified forms?

• Experimental design:

  • Perform 2D gel electrophoresis to separate isoforms.

  • Use phosphorylation-specific antibodies in parallel.

  • Key finding: The [EPR23821-101] clone detects full-length SUFU at 53 kDa but shows no cross-reactivity with truncated forms in KO models .

How to design a functional study linking SUFU antibody reactivity to Hedgehog pathway activity?

• Integrated workflow:

  • Co-immunoprecipitation: Pair SUFU antibody with Gli1/2 antibodies.

  • Quantitative readouts:

    • Measure nuclear-cytoplasmic Gli ratios via subcellular fractionation.

    • Correlate SUFU-Gli binding efficiency with pathway activation (e.g., qPCR of PTCH1).

Why does SUFU antibody show weak/no signal in certain cancer cell lines (e.g., LNCaP)?

• Hypothesis-driven analysis:

  • Epigenetic silencing: Check SUFU promoter methylation status.

  • Proteasomal degradation: Treat cells with MG-132 (proteasome inhibitor) for 6 hrs before lysis.

  • Alternative isoforms: Design primers to amplify SUFU transcripts for splice variant analysis.

How to address nonspecific bands in SUFU Western blots?

• Mitigation strategies:

  Band Size (kDa)	Likely Cause	Solution
  ~70	Protein aggregation	Add fresh β-mercaptoethanol
  ~40	Cross-reactive protein	Pre-adsorb antibody with KO lysate