FBXO30 Antibody

What is the optimal fixation method for immunostaining FBXO30 in tissue sections?

For effective immunostaining of FBXO30 in tissue sections, a multistep approach is recommended. Begin with deparaffinization and rehydration using xylene and ethanol gradients. Antigen retrieval is crucial and should be performed using 10 mM sodium citrate buffer (pH 6.0) at 95-100°C for 20-30 minutes. Permeabilize sections with 0.3% Triton X-100 in 10 mM Tris-HCl buffer for 30 minutes, followed by blocking with 2% bovine serum albumin (BSA) for 60 minutes. Incubate with primary FBXO30 antibody diluted in 10 mM Tris-HCl buffer containing 2% BSA at 4°C overnight, followed by appropriate secondary antibody incubation for 2-4 hours at room temperature. Counterstain nuclei with DAPI and mount using an anti-fade mounting medium .

How can I validate the specificity of an FBXO30 antibody for my research?

Antibody validation requires a multi-pronged approach. First, perform Western blotting using both recombinant FBXO30 protein and tissue/cell lysates known to express FBXO30, checking for a single band at the expected molecular weight (~30-35 kDa). Second, include a positive control (cells overexpressing FBXO30) and a negative control (FBXO30 knockout tissues or cells with FBXO30 silenced by shRNA). Third, perform immunoprecipitation followed by mass spectrometry to confirm that the antibody pulls down authentic FBXO30. Fourth, use immunofluorescence with blocking peptides to demonstrate specificity. Finally, parallel testing with at least two distinct antibodies targeting different epitopes of FBXO30 should yield similar results. Analysis of FBXO30 knockout mice tissues, which should show absence of staining, provides the gold standard for antibody validation .

What are the key experimental controls needed when studying FBXO30-substrate interactions?

When investigating FBXO30-substrate interactions, several critical controls are necessary. First, include negative controls using structurally similar F-box proteins (such as other FBXO family members) to confirm specificity. Second, perform reciprocal co-immunoprecipitation experiments (pull down with anti-FBXO30 and probe for the substrate, then pull down with anti-substrate and probe for FBXO30) as demonstrated with FBXO30 and EG5 interaction. Third, include proteasome inhibitor controls (e.g., MG132) to stabilize transient interactions. Fourth, utilize truncation mutants of both FBXO30 and the substrate to map interaction domains, as shown with the C-terminal region of EG5 (AA812-1052) being the interaction site with FBXO30. Fifth, confirm the functional consequences of the interaction through ubiquitination assays with recombinant E1, E2, FBXO30, substrate, and ubiquitin, followed by detection with anti-polyubiquitin antibodies .

What cell fractionation techniques are effective for detecting subcellular localization of FBXO30?

For effective subcellular localization of FBXO30, a differential centrifugation approach is recommended. Begin with gentle cell lysis using a hypotonic buffer containing protease inhibitors. Centrifuge at 1,000g for 10 minutes to separate nuclei (pellet). Further centrifuge the supernatant at 10,000g for 15 minutes to pellet mitochondria, followed by 100,000g for 1 hour to separate microsomes (pellet) from cytosol (supernatant). For more detailed fractionation, use density gradient centrifugation with sucrose or Percoll. Confirm fraction purity using markers: lamin A/C for nuclei, cytochrome c for mitochondria, calnexin for ER, and GAPDH for cytosol. For FBXO30 specifically, analyze fractions by Western blotting, with particular attention to nuclear and cytoplasmic fractions, as F-box proteins often shuttle between these compartments depending on cell cycle stage and substrate availability .

How can researchers distinguish between FBXO30's roles in different ubiquitination pathways targeting RARγ versus EG5?

Distinguishing between FBXO30's distinct ubiquitination targets requires carefully designed experiments that isolate each pathway. For RARγ pathway analysis, use cell systems where EG5 is either silenced or mutated at FBXO30 binding sites (C-terminal region AA812-1052). Conversely, for EG5 pathway studies, use systems where RARγ is inactivated. Temporal analysis is crucial, as FBXO30-RARγ interaction predominates during neural development, while FBXO30-EG5 interaction is critical during cell cycle progression and mammopoiesis.

Create the following experimental matrix to isolate each pathway:

For biochemical distinction, use in vitro ubiquitination assays with site-specific mutants. The E3 ligase activity of FBXO30 toward RARγ results in suppression of BMP signaling, while its activity toward EG5 affects centrosome homeostasis and mitotic spindle formation. These distinct downstream effects serve as reliable readouts for pathway-specific activity .

What are the methodological considerations when analyzing FBXO30 expression in neural tube defect tissues?

Analysis of FBXO30 expression in neural tube defect (NTD) tissues requires specialized methodological approaches. Fresh tissue procurement is critical, with rapid fixation (within 30 minutes post-collection) to preserve protein integrity. For human NTD samples, ethical considerations and appropriate consent protocols must be established. Parallel analysis of retinol levels is essential, as high retinol conditions affect FBXO30 levels.

For comprehensive analysis, implement this multi-modal approach:

• Quantitative immunohistochemistry using validated FBXO30 antibodies with automated image analysis to ensure unbiased quantification

• Laser capture microdissection to isolate specific cell populations from the neural tube for protein and RNA extraction

• Western blotting with gradient gels to detect both full-length FBXO30 and potential isoforms or degradation products

• qRT-PCR with multiple reference genes validated for stability in neural tissues

• Analysis of BMP target gene expression (downstream readout of FBXO30-RARγ pathway)

When comparing NTD and control tissues, account for variables including gestational age, sample preservation time, and retinol levels. A scoring system incorporating both FBXO30 levels and downstream targets (BMP pathway genes) provides the most reliable assessment of FBXO30 functional status in NTD tissues .

How can researchers design experiments to determine if aberrant FBXO30 function contributes to mitotic spindle defects in cancer cells?

To investigate FBXO30's role in mitotic spindle defects in cancer cells, a comprehensive experimental design should include:

First, establish baseline FBXO30 and EG5 expression profiles across cancer cell lines using Western blotting, qRT-PCR, and immunofluorescence. Next, implement a CRISPR-Cas9 approach to generate FBXO30 knockout cancer cell lines alongside rescue lines expressing either wild-type FBXO30 or ligase-dead mutants.

For functional analysis, employ live-cell imaging with fluorescently tagged tubulin to track mitotic spindle dynamics in real-time. Quantify multipolar spindle formation, spindle pole fragmentation, and chromosome segregation errors. Perform immunofluorescence co-staining for FBXO30, EG5, and γ-tubulin to analyze centrosome amplification and spindle morphology.

To establish causality, manipulate EG5 levels through siRNA knockdown or EG5 inhibitors (such as monastrol) in both FBXO30-normal and FBXO30-deficient cells. If abnormal spindle phenotypes in FBXO30-deficient cells are normalized through EG5 reduction, this confirms FBXO30's regulatory role through EG5. Additionally, perform cell cycle synchronization experiments using double thymidine block to analyze FBXO30 and EG5 oscillation patterns during mitosis, with specific attention to G2/M transition where aberrant EG5 activity leads to multipolar spindles and genomic instability .

What approaches can be used to investigate the tissue-specific functions of FBXO30 in mammopoiesis versus neural development?

For mammopoiesis studies, analyze:

• Mammary epithelial cell populations using flow cytometry with markers CD24, CD29, CD49f, and Sca1

• Mammosphere formation assays to assess stem cell function

• Immunohistochemistry for lineage markers (Keratin 5 for myoepithelial cells)

• Mitotic spindle analysis in mammary epithelial cells using β-tubulin and DAPI staining

• Quantification of EG5 protein levels and cell cycle distribution

For neural development studies, focus on:

• BMP signaling pathway components and their expression levels

• RARγ degradation kinetics in neural progenitor cells

• Neural tube closure in embryonic development

• Retinol and retinoic acid level measurements

• Neural progenitor cell proliferation and differentiation markers

Cross-tissue analysis should include ubiquitination assays comparing FBXO30's preferential substrates (EG5 versus RARγ) in each tissue type, and rescue experiments using tissue-specific expression of either substrate to determine if the phenotypes are reversible .

What are the technical considerations for developing phospho-specific antibodies to study FBXO30 regulation?

Developing phospho-specific antibodies for FBXO30 regulation studies requires careful consideration of multiple technical factors. First, perform in silico analysis using phosphorylation prediction algorithms (NetPhos, GPS, PhosphoSitePlus) to identify potential phosphorylation sites on FBXO30. Validate these sites using mass spectrometry following immunoprecipitation of FBXO30 from cells treated with phosphatase inhibitors.

For antibody development, synthetic phosphopeptides should span 10-15 amino acids centered on the phosphorylation site, with the following considerations:

• Include a terminal cysteine for carrier protein conjugation if not naturally present

• Ensure peptide solubility and minimize secondary structure formation

• Create paired phosphorylated and non-phosphorylated peptides for screening

• Consider multiple host species to maximize application versatility

Antibody validation should include:

• ELISA testing against phosphorylated versus non-phosphorylated peptides

• Western blotting with lysates from cells treated with kinase activators/inhibitors

• Competition assays with free phosphopeptides

• Testing on FBXO30 knockout samples (negative control)

• Testing on samples treated with lambda phosphatase

For understanding FBXO30 regulation, particularly important is examining how phosphorylation affects FBXO30-substrate interactions. Unlike EG5, where phosphorylation at T927 or S1040 doesn't affect FBXO30-mediated ubiquitination, FBXO30's own phosphorylation state may determine substrate specificity between RARγ and EG5 in different cellular contexts .

How can researchers effectively design cell synchronization experiments to study FBXO30 dynamics during the cell cycle?

Cell synchronization experiments for studying FBXO30 dynamics require precise timing and multiple synchronization methods for comprehensive analysis. Implement a double thymidine block protocol (2 mM thymidine for 18 hours, release for 9 hours, second thymidine treatment for 17 hours) to arrest cells at G1/S boundary. After release, collect samples every 2 hours for 24 hours to cover a complete cell cycle.

Alternative synchronization approaches include:

• Nocodazole treatment (100 ng/mL for 12-16 hours) for M-phase arrest

• Serum starvation (0.1% serum for 48 hours) for G0/G1 arrest

• RO-3306 (10 μM for 18 hours) for G2/M arrest

For each time point, analyze:

• FBXO30 protein levels by Western blotting

• FBXO30 subcellular localization by immunofluorescence

• FBXO30-substrate interactions by co-immunoprecipitation

• Ubiquitination status of FBXO30 targets

• Cell cycle distribution by flow cytometry with propidium iodide staining

Critical controls include asynchronous cell populations and validation of synchronization efficiency using established cell cycle markers (cyclin B1, phospho-histone H3). Based on findings from EG5 studies, pay particular attention to G2/M transition, where FBXO30-mediated regulation appears most critical for normal mitotic spindle formation and chromosome segregation .

What are the most reliable techniques for detecting FBXO30-mediated ubiquitination of target proteins in vitro and in vivo?

Detecting FBXO30-mediated ubiquitination requires different approaches for in vitro and in vivo scenarios. For in vitro ubiquitination:

• Reconstitute the complete ubiquitination cascade using purified components:

  • Recombinant E1 ubiquitin-activating enzyme

  • E2 ubiquitin-conjugating enzyme

  • FBXO30 immunoprecipitated from mammalian cells or recombinant

  • Purified substrate (RARγ or EG5)

  • Ubiquitin (consider using tagged versions for easier detection)

  • ATP and buffer components

• Incubate the reaction at 30°C for 1-2 hours and analyze by SDS-PAGE followed by immunoblotting with anti-ubiquitin antibodies.

For in vivo ubiquitination:

• Treat cells with proteasome inhibitors (MG132, 10 μM for 4-6 hours) to prevent degradation of ubiquitinated proteins

• Perform denaturing immunoprecipitation to disrupt protein-protein interactions:

  • Lyse cells in buffer containing 1% SDS

  • Dilute lysate to 0.1% SDS before immunoprecipitation

  • Immunoprecipitate the substrate of interest

  • Analyze by Western blotting with anti-ubiquitin antibodies

• Alternative approach using His-tagged ubiquitin:

  • Transfect cells with His-ubiquitin

  • Lyse cells under denaturing conditions

  • Purify ubiquitinated proteins using Ni-NTA resin

  • Detect specific substrates by immunoblotting

Controls should include FBXO30 knockout cells, substrate mutants lacking FBXO30 binding sites, and F-box domain mutants of FBXO30 that cannot form an active SCF complex .

How can researchers correlate FBXO30 expression levels with clinical outcomes in developmental disorders?

To correlate FBXO30 expression with clinical outcomes in developmental disorders, researchers should implement a multi-layer analysis approach. Begin with tissue microarray construction from well-characterized patient cohorts with detailed clinical data and long-term follow-up. Perform immunohistochemistry with validated FBXO30 antibodies, using automated quantitative analysis (AQUA) to generate objective expression scores.

For neural tube defects specifically, establish a comprehensive assessment matrix:

Statistical analysis should include multivariate models adjusting for confounding factors such as gestational age, maternal nutrition, and genetic background. Longitudinal studies with consistent follow-up intervals are essential for correlating early FBXO30 expression patterns with long-term developmental outcomes. Case-control studies comparing NTD samples with matched controls allow for identification of FBXO30 expression thresholds that predict adverse outcomes .

What is the recommended workflow for analyzing FBXO30 knockout phenotypes in various model systems?

A comprehensive workflow for analyzing FBXO30 knockout phenotypes across model systems should follow a systematic approach from molecular to organismal levels. Begin with validation of knockout efficiency through genomic PCR, Western blotting, and immunostaining to confirm absence of FBXO30 protein.

For cellular phenotyping:

• Analyze cell cycle distribution using flow cytometry with propidium iodide staining

• Examine mitotic spindle formation through immunofluorescence for β-tubulin

• Quantify centrosome numbers using γ-tubulin staining

• Assess genomic stability through karyotyping and micronuclei formation analysis

• Measure proliferation rates using BrdU incorporation assays

For tissue-specific analysis:

• In mammary tissue:

  • Flow cytometry for epithelial cell populations (CD24+CD29lo luminal and CD24+CD29hi myoepithelial)

  • Mammosphere formation assays

  • Whole-mount analysis of ductal development

  • Immunohistochemistry for lineage markers

• In neural tissue:

  • Neural tube closure assessment in embryos

  • BMP signaling target gene expression

  • Neuroprogenitor proliferation and differentiation markers

For rescue experiments, introduce either wild-type FBXO30 or target substrate modulators (EG5 inhibitors or shRNA) to determine phenotype reversibility. Establish clear quantitative metrics for each phenotype to enable statistical comparison across experiments and between different model systems .

How should researchers approach antibody selection for multiplex immunofluorescence studies involving FBXO30 and its target proteins?

Successful multiplex immunofluorescence studies involving FBXO30 and its target proteins require careful antibody selection and validation. Begin with comprehensive antibody screening against single targets in positive control tissues before attempting multiplexing. Consider these key factors:

• Host species compatibility:

  • Select primary antibodies raised in different host species (e.g., rabbit anti-FBXO30, mouse anti-EG5, rat anti-RARγ)

  • If using same-species antibodies, employ sequential staining with complete HRP inactivation between rounds

• Antibody format selection:

  • For direct detection: Pre-conjugated primary antibodies with non-overlapping fluorophores

  • For indirect detection: Carefully matched secondary antibodies with minimal cross-reactivity

• Signal amplification considerations:

  • For low abundance proteins: Tyramide signal amplification (TSA)

  • For co-localization studies: Standard indirect immunofluorescence to minimize spatial distortion

• Multiplexing panel design:

  Target	Abundance	Recommended Approach	Signal Separation
  FBXO30	Low-moderate	Rabbit mAb + TSA	488 nm (green)
  EG5/KIF11	Moderate	Mouse mAb + standard IF	555 nm (red)
  RARγ	Low	Goat pAb + TSA	647 nm (far red)
  Cellular context marker	Variable	Directly conjugated	405 nm (blue)

• Validation controls:

  • Single staining controls for each antibody

  • Fluorescence minus one (FMO) controls

  • Peptide competition controls

  • FBXO30 knockout tissues as negative control

For optimal visualization of protein interactions, complement standard confocal microscopy with super-resolution techniques such as structured illumination microscopy (SIM) or stochastic optical reconstruction microscopy (STORM) for detailed co-localization analysis at the subcellular level .

What are the emerging techniques for studying dynamic interactions between FBXO30 and its substrates in live cells?

Several cutting-edge technologies are emerging for real-time analysis of FBXO30-substrate interactions in living cells. Proximity ligation assays (PLA) offer visualization of protein interactions within 40 nm distance, providing spatial information about FBXO30-substrate complexes. For real-time dynamics, bimolecular fluorescence complementation (BiFC) can be employed, where split fluorescent proteins fused to FBXO30 and its substrate reconstitute fluorescence upon interaction.

Förster resonance energy transfer (FRET) approaches using FBXO30 and substrates tagged with compatible fluorophores (e.g., CFP-FBXO30 and YFP-EG5) enable quantitative assessment of protein-protein interactions with high temporal resolution during cell cycle progression. Lattice light-sheet microscopy combined with these fluorescent reporters offers superior spatial and temporal resolution with minimal phototoxicity for extended imaging.

For substrate fate tracking, fluorescent timer proteins fused to FBXO30 targets provide color-coded information about protein age and turnover rates. CRISPR-based gene tagging with split fluorescent proteins enables visualization of endogenous protein interactions without overexpression artifacts.

Correlative light and electron microscopy (CLEM) can bridge the resolution gap between fluorescence microscopy and ultrastructural analysis, particularly valuable for studying FBXO30's role in mitotic spindle formation and centrosome dynamics. Mass spectrometry imaging (MSI) is emerging as a label-free approach to map protein modifications, including ubiquitination patterns, across cellular compartments .

How can computational approaches improve understanding of FBXO30 substrate recognition and specificity?

Computational approaches offer powerful tools for unraveling FBXO30 substrate recognition mechanisms. Structural modeling using homology-based techniques can predict the three-dimensional conformation of FBXO30's substrate-binding domain based on known F-box protein structures. Molecular docking simulations can then identify potential interaction interfaces between FBXO30 and substrates like RARγ and EG5.

Machine learning algorithms trained on known E3 ligase-substrate pairs can predict additional FBXO30 substrates by analyzing protein features such as degron motifs, disorder regions, and post-translational modification sites. Network analysis integrating protein-protein interaction data, co-expression patterns, and evolutionary conservation can reveal substrate candidates within FBXO30's functional pathways.

Molecular dynamics simulations offer insights into the conformational changes of FBXO30 upon substrate binding and how these changes affect ubiquitin transfer. These simulations can also elucidate how phosphorylation of either FBXO30 or its substrates alters binding affinity and specificity.

Text mining approaches can identify potential FBXO30-substrate relationships from the scientific literature that might have been overlooked. Combined with systems biology approaches modeling ubiquitin-proteasome dynamics, these computational methods provide testable hypotheses about FBXO30's role in maintaining proteostasis across different tissue contexts and developmental stages .

What are the recommended protocols for generating and validating FBXO30 knockout and knockdown models?

Generating reliable FBXO30 knockout and knockdown models requires careful selection of appropriate techniques based on experimental goals. For complete knockout models, CRISPR-Cas9 genome editing offers the most definitive approach. Design sgRNAs targeting early exons of FBXO30, preferably exon 1, to ensure complete protein elimination. Use multiple guide RNAs to increase editing efficiency, and screen clones by genomic PCR, Sanger sequencing, and Western blotting.

For conditional knockout models critical for tissue-specific studies:

• Generate FBXO30-floxed mice with loxP sites flanking critical exons

• Cross with tissue-specific Cre lines (e.g., MMTV-Cre for mammary, Nestin-Cre for neural)

• Validate recombination efficiency by tissue-specific genomic PCR and immunoblotting

For transient knockdown applications:

• siRNA approach: Design 3-4 siRNAs targeting different regions of FBXO30 mRNA

• shRNA approach: Use inducible systems (Tet-On/Off) for temporal control

• Validation criteria: >80% reduction in FBXO30 protein levels within 48-72 hours

• Critical controls: Non-targeting siRNA/shRNA and rescue with siRNA-resistant FBXO30

Regardless of the approach, comprehensive validation is essential:

• Genomic verification: PCR and sequencing of targeted region

• Transcript analysis: RT-PCR and RNA-seq to confirm absence of FBXO30 mRNA

• Protein validation: Western blotting and immunostaining with antibodies targeting different epitopes

• Functional validation: Analysis of substrate accumulation (RARγ, EG5)

• Phenotypic confirmation: Assessment of cellular processes known to be regulated by FBXO30 (mitotic spindle formation, BMP signaling) .

What quality control measures should be implemented when using commercial FBXO30 antibodies for different applications?

Implementing robust quality control measures for commercial FBXO30 antibodies is essential for reliable experimental outcomes. For any new antibody lot, perform Western blotting validation using positive controls (tissues known to express FBXO30 such as mammary epithelial cells) and negative controls (FBXO30 knockout tissues or cells with FBXO30 knocked down by siRNA/shRNA). Verify the antibody detects a single band of appropriate molecular weight (~30-35 kDa).

Application-specific quality control measures:

For immunohistochemistry/immunofluorescence:

• Test multiple antibody dilutions to determine optimal signal-to-noise ratio

• Include absorption controls using blocking peptides

• Compare staining patterns across multiple FBXO30 antibodies targeting different epitopes

• Include FBXO30 knockout tissues as negative controls

• Validate subcellular localization against known patterns

For immunoprecipitation:

• Verify pull-down efficiency by comparing input, unbound, and immunoprecipitated fractions

• Confirm co-immunoprecipitation of known interaction partners (Skp1, Cul1)

• Perform reverse immunoprecipitation with antibodies against known partners

• Test specificity using structurally similar F-box proteins

For flow cytometry:

• Establish appropriate fixation and permeabilization protocols

• Perform fluorescence-minus-one controls

• Validate with cells expressing fluorescently tagged FBXO30