FBXO3 Antibody

What are the common applications for FBXO3 antibodies in research?

FBXO3 antibodies are primarily used in the following experimental applications:

• Western Blot (WB): Recommended dilution ranges from 1:500-1:3000, with optimal results at sample-dependent concentrations

• Co-Immunoprecipitation (CoIP): For studying FBXO3 protein interactions with substrates like FBXL2

• Immunohistochemistry-Paraffin (IHC-P): For detection in fixed tissue samples

• Immunocytochemistry/Immunofluorescence (ICC/IF): For subcellular localization studies

Different applications require specific sample preparation techniques and antibody concentrations for optimal results .

How should researchers validate the specificity of FBXO3 antibodies?

Validation should include:

• Knockout/Knockdown controls: Compare results with FBXO3 knockout/knockdown cells as negative controls

• Molecular weight verification: Confirm detection at the expected molecular weight (48-50 kDa observed; 55 kDa calculated)

• Cross-reactivity testing: Test antibody reactivity across different species (human, mouse, rat)

• Epitope mapping: Verify recognition of the target epitope region (antibodies like ab224603 target amino acids 250-400)

What is the recommended protocol for using FBXO3 antibodies in Western blot analysis?

For optimal Western blot results with FBXO3 antibodies:

• Sample preparation:

  • Lyse cells in appropriate buffer with protease inhibitors

  • Load 20-40 μg of total protein per lane

• Electrophoresis and transfer:

  • Separate proteins on 10% SDS-PAGE gel

  • Transfer to PVDF membrane at 100V for 60-90 minutes

• Antibody incubation:

  • Block membrane with 5% non-fat milk in TBST for 1 hour

  • Incubate with primary FBXO3 antibody (1:500-1:3000 dilution) overnight at 4°C

  • Wash 3x with TBST

  • Incubate with HRP-conjugated secondary antibody for 1 hour at room temperature

• Detection:

  • Expected band size: 48-50 kDa

  • Positive control tissues: HeLa cells, HepG2 cells

How can researchers effectively study FBXO3-substrate interactions using antibody-based approaches?

To investigate FBXO3-substrate interactions:

• Co-immunoprecipitation strategy:

  • Treat cells with proteasome inhibitor (MG132, 10 μM) for 6-10 hours before harvesting

  • Immunoprecipitate using anti-FBXO3 antibody coupled to Protein A/G beads

  • Detect potential substrates in the immunoprecipitated complex

• In vitro binding assays:

  • Purify FBXO3 using immunoprecipitation with HA-tagged FBXO3

  • Incubate with potential substrate proteins

  • Analyze binding using Western blot

• Mutational analysis:

  • Compare binding affinity between wild-type FBXO3 and domain mutants (ΔF-box or ΔApaG)

  • Determine which domains are critical for specific substrate interactions

For example, studies have shown that the ApaG domain (amino acids 294-303, loop 1) is critical for FBXL2 binding, whereas ΔNp63α degradation requires the F-box domain but not the ApaG domain .

How can researchers design experiments to investigate the role of FBXO3 in inflammatory pathways?

Experimental design for studying FBXO3 in inflammation:

• Cell culture models:

  • Stimulate human blood mononuclear cells with lipopolysaccharide (LPS)

  • Compare cytokine production in FBXO3 knockdown vs. control conditions

  • Measure cytokine secretion by ELISA or multiplex assays

• Genetic manipulation approaches:

  • Generate stable cell lines expressing:

    • Wild-type FBXO3

    • FBXO3 ΔF-box (E3 ligase-defective mutant)

    • FBXO3 ΔApaG (FBXL2 binding-defective mutant)

  • Compare effects on TRAF protein levels and cytokine secretion

• Pharmacological inhibition:

  • Treat cells with BC-1215 or other benzathine-based FBXO3 inhibitors

  • Determine IC50 values for inhibition of FBXO3-FBXL2 interaction

  • Measure downstream effects on TRAF stability and cytokine production

What are the recommended controls when studying FBXO3-mediated protein degradation?

Essential controls for FBXO3-mediated degradation studies:

• Proteasome inhibition controls:

  • Compare substrate levels with/without MG132 (10 μM, 6-10 hours)

  • Verify accumulation of ubiquitinated proteins in presence of MG132

• Protein half-life determination:

  • Cycloheximide chase assay (100 μg/ml) with timepoints at 0, 1, 4, and 8 hours

  • Compare substrate half-life in FBXO3 knockdown vs. control conditions

• Ubiquitination specificity controls:

  • Include negative controls:

    • FBXO3 ΔF-box mutant (E3 ligase deficient)

    • Ubiquitin lysine mutants (K48 vs. K63 linkages)

  • In vitro ubiquitination assay components:

    • Recombinant E1 (1.5 ng/μl)

    • E2 enzymes (UbcH5a and UbcH7, 10 ng/μl each)

    • ATP (2 mM)

    • Ubiquitin (1 mg/ml)

What are common issues when using FBXO3 antibodies in co-immunoprecipitation experiments?

Common challenges and solutions:

• Weak interaction detection:

  • Pre-treat cells with proteasome inhibitor (MG132, 10 μM) for 6-10 hours

  • Use cell-permeable crosslinkers for transient interactions

  • Reduce washing stringency (use 0.1% instead of 0.5% Triton X-100)

• High background issues:

  • Increase blocking duration (1 hour minimum with 5% BSA)

  • Use more stringent washing buffers

  • Include pre-clearing step with protein A/G beads

• Domain-specific interactions:

  • When studying FBXO3-FBXL2 interaction, the ApaG domain is critical

  • Loop 1 (residues 294-303) of the ApaG domain is especially important

  • For studying interactions with other substrates like ΔNp63α, focus on the F-box domain

Protocol optimization table:

How can researchers distinguish between different FBXO3 isoforms using antibodies?

Strategies for isoform discrimination:

• Epitope mapping approach:

  • Select antibodies targeting regions that differ between isoforms

  • Verify epitope recognition using deletion mutants in overexpression systems

• Molecular weight analysis:

  • Full-length FBXO3: Expected 55 kDa (calculated), observed 48-50 kDa

  • Verify band pattern using FBXO3 knockout controls

  • Run high-resolution SDS-PAGE (8-10%) to separate closely-sized isoforms

• Functional validation:

  • Use isoform-specific siRNAs to selectively knockdown specific variants

  • Confirm antibody specificity by observing selective band reduction

What are the optimal conditions for immunofluorescence detection of FBXO3?

For successful immunofluorescence with FBXO3 antibodies:

• Cell preparation:

  • Culture cells on appropriate substrates (e.g., Collagen I coated coverslips)

  • Fix with 4% formaldehyde for 30 minutes at room temperature

  • Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes

• Antibody incubation:

  • Block with 5% BSA in PBS for 1 hour

  • Dilute primary antibody in 1% BSA/PBS (typically 1:100-1:500)

  • Incubate at 4°C overnight

  • Use appropriate fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488 or 594)

  • Incubate for 2 hours at room temperature

• Controls and co-staining:

  • Include FBXO3 knockdown negative control

  • Consider co-staining with cellular compartment markers

  • For substrate co-localization studies, double immunofluorescence can visualize potential interaction sites

How does FBXO3 structural conformation affect its antibody epitope accessibility?

The structural features of FBXO3 that impact antibody recognition:

• Domain architecture considerations:

  • The ApaG domain (C-terminal region) forms a compact structure consisting of seven β-strands

  • Structural differences between human and bacterial ApaG domains include an additional N-terminal β-strand in human FBXO3

  • Antibodies targeting flexible loop regions (particularly loop 1, residues 294-303) may have variable accessibility depending on binding partners

• Post-translational modification effects:

  • Phosphorylation states may alter epitope accessibility

  • Interaction with substrates like FBXL2 may mask certain epitopes

  • Consider native vs. denatured detection methods when selecting antibodies

• Structure-function implications:

  • X-ray crystallography data at 2.0 Å resolution shows the FBXO3 ApaG domain has an IgG/Fibronectin III-type fold

  • Antibodies recognizing this region should be validated in both western blot and immunoprecipitation applications

How can researchers resolve contradictory findings when studying FBXO3-mediated substrate degradation?

Resolving conflicting experimental results:

• Substrate-specific considerations:

  • Different FBXO3 substrates utilize distinct recognition mechanisms:

    • FBXL2 degradation requires the ApaG domain

    • ΔNp63α degradation requires the F-box domain but not the ApaG domain

  • Confirm which domain is involved in your specific substrate interaction

• Experimental context differences:

  • Cell type-specific effects: Validate findings across multiple cell lines

  • Stimulation conditions: LPS treatment may alter FBXO3 activity

  • Temporal dynamics: Consider kinetics of substrate degradation

• Technical validation approach:

  • Verify antibody specificity against your substrate

  • Use multiple siRNAs targeting different regions of FBXO3

  • Employ complementary techniques (in vitro and in vivo ubiquitination)

What are the methodological considerations when studying FBXO3 in different disease models?

Disease-specific methodological approaches:

• Inflammatory disorders:

  • Models: LPS-induced inflammation, viral pneumonia, septic shock, colitis

  • Readouts: Cytokine production (TNF-α, IL-1β, IL-6)

  • Intervention: FBXO3 inhibitors (benzathine derivatives) targeting the ApaG domain

  • Controls: Compare with established anti-inflammatory agents

• Cancer models:

  • Tissue-specific considerations: Different expression patterns in leukemia, pituitary adenoma, oral squamous cell carcinoma

  • Signaling pathway analysis: TGF-β signaling, NF-κB pathway activation

  • Prognostic correlation: Correlate FBXO3 expression with patient outcomes

  • Intervention: siRNA knockdown, CRISPR/Cas9 knockout, small molecule inhibitors

• Methodological adaptations:

  • Cell line selection based on endogenous FBXO3 expression levels

  • Timing of analysis after stimulus/inhibitor application

  • Consideration of compensatory mechanisms in chronic models

How might emerging structural biology techniques enhance FBXO3 antibody development and application?

Cutting-edge structural approaches:

• Cryo-electron microscopy applications:

  • Visualize FBXO3 in complex with SCF components and substrates

  • Determine conformational changes upon substrate binding

  • Identify novel epitopes for antibody development

• Structure-guided antibody design:

  • Target specific functional domains (F-box vs. ApaG)

  • Develop conformation-specific antibodies to detect active vs. inactive FBXO3

  • Create antibodies that selectively recognize FBXO3 in complex with specific substrates

• Technical considerations:

  • The crystal structure of FBXO3 ApaG domain (2.0 Å resolution) reveals key structural features

  • Structural alignments with bacterial ApaG proteins show conservation despite sequence divergence

  • Integrating structural data can guide epitope selection for higher specificity antibodies

What methodological approaches should researchers consider when studying FBXO3 in the context of therapeutic development?

Research strategies for therapeutic applications:

• Inhibitor development methodology:

  • Structure-based design targeting the ApaG domain

  • Screening approaches:

    • Protein interaction assays (Fbxo3-Fbxl2 binding)

    • IC50 determination (10^-11 to 10^-4 M concentration range)

    • Virtual screening using LigandFit (Discovery Studio)

• Target validation techniques:

  • In vitro validation using purified proteins

  • Cellular models with cytokine readouts

  • Animal models of inflammation (viral pneumonia, septic shock, colitis)

  • Pharmacokinetic and pharmacodynamic studies

• Biomarker development:

  • FBXO3 expression and activity measurement

  • Substrate levels (FBXL2, TRAFs, etc.)

  • Downstream effectors (cytokine profiles)

  • Correlation with disease severity

How can researchers combine genomic and proteomic approaches to better understand FBXO3 function across different cellular contexts?

Integrated omics strategies:

• Multi-omics experimental design:

  • RNA-seq to identify FBXO3-dependent gene expression

  • Proteomics to identify novel substrates and interaction networks

  • Phosphoproteomics to map regulatory post-translational modifications

  • ChIP-seq to identify transcriptional targets affected by FBXO3 activity

• Cellular context considerations:

  • Compare findings across multiple cell types

  • Analyze under both basal and stimulated conditions

  • Consider time-course experiments to capture dynamic changes

• Data integration approaches:

  • Network analysis to identify functional modules

  • Machine learning to predict novel substrates

  • Pathway enrichment to understand biological processes regulated by FBXO3

  • Cross-reference with disease-associated databases to identify therapeutic opportunities