FBL15 Antibody

What is FBXL15 and what cellular functions does it regulate?

FBXL15, also known as FBXO37, belongs to the FBXL15 family of proteins. It functions primarily in the G2/M transition of the cell cycle and participates in cellular protein metabolic processes. Research indicates that FBXL15 is also involved in the positive regulation of bone development . As an F-box protein, it likely forms part of the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex, which is responsible for protein ubiquitination and subsequent degradation by the proteasome. This positions FBXL15 as a potential regulator of protein turnover in various cellular contexts.

What applications can FBXL15 antibodies be used for in research?

FBXL15 antibodies have been validated for multiple research applications:

For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 may serve as an alternative . The antibody shows reactivity with human samples and has been successfully used to detect a protein of approximately 33 kDa, which corresponds to the calculated molecular weight of FBXL15.

How should FBXL15 antibodies be stored to maintain optimal performance?

For optimal preservation of antibody function, FBXL15 antibodies should be stored at -20°C where they remain stable for one year after shipment. The antibody is typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . Importantly, aliquoting is unnecessary for storage at -20°C. Some preparations, particularly those in smaller volumes (e.g., 20μl), may contain 0.1% BSA as a stabilizer. When working with the antibody, avoid repeated freeze-thaw cycles as these can degrade antibody performance over time.

How can I confirm the specificity of my FBXL15 antibody for advanced research applications?

Confirming antibody specificity is crucial for reliable research outcomes. For FBXL15 antibody validation, implement the following comprehensive approach:

• Knockout/knockdown validation: Generate FBXL15 knockout or knockdown cell lines using CRISPR-Cas9 or siRNA technology. Compare Western blot signals between wildtype and knockout/knockdown samples to confirm specificity.

• Overexpression studies: Transfect cells with FBXL15-expressing vectors and verify increased signal intensity on Western blots.

• Immunoprecipitation followed by mass spectrometry: Perform IP with the FBXL15 antibody and analyze pulldown products by MS to confirm presence of FBXL15 and known interacting partners.

• Peptide competition assay: Pre-incubate the antibody with the immunogenic peptide used to generate it (FBXL15 fusion protein Ag14949) before application. Signal disappearance indicates specificity .

• Multiple antibody verification: Compare results using different antibodies targeting distinct epitopes of FBXL15.

This methodical validation process ensures confidence in subsequent experimental interpretations, particularly for mechanistic studies of FBXL15 function.

What experimental considerations are important when studying FBXL15's role in the cell cycle?

When investigating FBXL15's functions in cell cycle regulation, particularly the G2/M transition , consider these methodological approaches:

• Cell synchronization: Implement synchronization protocols (e.g., double thymidine block or nocodazole treatment) to enrich cell populations at specific cycle phases. Monitor FBXL15 expression and localization throughout the cell cycle using immunofluorescence and Western blotting.

• Co-immunoprecipitation studies: Identify cell cycle-specific interaction partners of FBXL15 by performing co-IP experiments at different cell cycle stages. This may reveal temporal regulation of substrate recognition.

• Ubiquitination assays: As an F-box protein, FBXL15 likely targets proteins for ubiquitination. Use in vitro and in vivo ubiquitination assays to identify substrates relevant to cell cycle regulation.

• Phosphorylation analysis: Examine whether FBXL15 is post-translationally modified during cell cycle progression, as phosphorylation often regulates F-box protein activity.

• Live-cell imaging: Implement live-cell imaging with fluorescently tagged FBXL15 to track its dynamics during cell cycle progression.

• Single-cell analysis: Consider single-cell approaches to address heterogeneity in FBXL15 expression and function across a cell population.

These approaches enable comprehensive investigation of FBXL15's mechanistic role in cell cycle regulation beyond mere correlation studies.

How can I analyze potential cross-reactivity of FBXL15 antibodies with other F-box family proteins?

F-box proteins share structural similarities, particularly in the F-box domain, which may lead to cross-reactivity issues. To assess and address potential cross-reactivity of FBXL15 antibodies:

• Sequence alignment analysis: Perform bioinformatic alignment of the immunogen sequence (FBXL15 fusion protein Ag14949) against other F-box family proteins to identify regions of homology that might cause cross-reactivity.

• Overexpression panel: Generate a panel of cells overexpressing different F-box proteins (particularly closely related FBXL family members) and test the antibody against this panel by Western blotting or immunofluorescence.

• Antibody absorption tests: Pre-absorb the antibody with recombinant proteins from related F-box family members to determine if this eliminates specific signal.

• Epitope mapping: If resources permit, determine the exact epitope recognized by the antibody using epitope mapping techniques, which allows more precise prediction of cross-reactivity potential.

• Biophysics-informed modeling: Apply computational approaches similar to those used for antibody specificity prediction to assess potential cross-reactivity based on binding energetics.

What optimization steps are necessary for successful Western blot detection of FBXL15?

For optimal Western blot detection of FBXL15 (observed molecular weight: 33 kDa) , follow this methodological approach:

• Sample preparation:

  • Use strong lysis buffers containing protease inhibitors

  • Include phosphatase inhibitors if phosphorylated forms are of interest

  • Maintain samples at 4°C during preparation to prevent degradation

• Antibody dilution optimization:

  • Begin with manufacturer's recommended range (1:200-1:1000)

  • Perform a dilution series (e.g., 1:200, 1:500, 1:1000) to determine optimal signal-to-noise ratio

  • Consider extended incubation at 4°C overnight for lower concentrations

• Blocking optimization:

  • Test multiple blocking agents (5% non-fat milk, 5% BSA)

  • BSA is often preferred for phospho-specific detection

• Positive control inclusion:

  • Include MDA-MB-453s cell lysate as positive control

  • Consider including FBXL15-overexpressing cells

• Transfer optimization:

  • For the 33 kDa FBXL15 protein, standard semi-dry or wet transfer methods are suitable

  • Transfer time: 60-90 minutes at 100V or 25V overnight

• Detection system selection:

  • For low abundance detection, consider enhanced chemiluminescence (ECL) or fluorescence-based systems

  • Longer exposure times may be necessary depending on expression levels

• Stripping and reprobing considerations:

  • Mild stripping buffers are recommended if membrane reuse is necessary

  • Validate complete stripping before reprobing

This systematic approach allows for reproducible and specific detection of FBXL15 in complex biological samples.

What are the key considerations for optimizing FBXL15 immunohistochemistry protocols?

Optimizing IHC protocols for FBXL15 detection requires careful attention to multiple parameters:

• Tissue preparation and fixation:

  • Fixation: 10% neutral buffered formalin for 24-48 hours

  • Paraffin embedding using standard protocols

  • Section thickness: 4-5 μm for optimal antibody penetration

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: Citrate buffer pH 6.0

  • Heat-induced epitope retrieval: 95-98°C for 15-20 minutes followed by 20-minute cooling

• Antibody dilution:

  • Initial testing range: 1:50-1:500

  • Perform titration experiments to determine optimal concentration

  • Include positive control tissue (human breast cancer tissue)

• Incubation conditions:

  • Primary antibody: Overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody: 30-60 minutes at room temperature

• Detection system selection:

  • For chromogenic detection: DAB (3,3'-diaminobenzidine) provides good contrast

  • For fluorescence: Choose fluorophores with minimal spectral overlap for co-localization studies

• Counterstaining optimization:

  • Hematoxylin counterstaining: 30-60 seconds for nuclear visualization

  • Adjust timing to prevent overstaining which may mask weak signals

• Controls:

  • Positive control: Human breast cancer tissue

  • Negative controls: Primary antibody omission and isotype controls

  • Competitive inhibition with immunizing peptide

• Signal amplification:

  • Consider tyramide signal amplification for low-abundance targets

  • Polymer-based detection systems can enhance sensitivity while reducing background

This comprehensive approach ensures reliable and reproducible FBXL15 detection in tissue sections for research applications.

How can multiplex immunofluorescence be used to study FBXL15 interactions with other proteins?

Multiplex immunofluorescence allows simultaneous visualization of FBXL15 and potential interaction partners in situ. Here's a methodological approach for studying FBXL15 protein interactions:

• Antibody panel design:

  • Select antibodies against FBXL15 and suspected interaction partners

  • Ensure antibodies are raised in different host species to avoid cross-reactivity

  • If using multiple rabbit antibodies, consider sequential tyramide signal amplification

• Fluorophore selection:

  • Choose fluorophores with minimal spectral overlap

  • Consider brightness and photostability characteristics

  • Typical combinations: DAPI (nuclei), Alexa Fluor 488, Cy3, Alexa Fluor 647

• Sample preparation optimization:

  • Cell fixation: 4% paraformaldehyde (10-15 minutes)

  • Permeabilization: 0.1-0.3% Triton X-100 (10 minutes)

  • Blocking: 5-10% normal serum from the species of secondary antibodies

• Antibody labeling sequence:

  • For standard co-staining: Apply primary antibodies simultaneously or sequentially

  • For sequential multiplex IF: Complete each round of staining followed by signal capture and antibody stripping

• Quantitative colocalization analysis:

  • Calculate Pearson's correlation coefficient or Manders' overlap coefficient

  • Implement intensity correlation analysis

  • Use object-based approaches for discrete structures

• Advanced visualization methods:

  • Super-resolution microscopy (STED, STORM, SIM) for nanoscale interaction analysis

  • Live-cell imaging with tagged proteins to monitor dynamic interactions

  • FRET or PLA (Proximity Ligation Assay) for direct interaction validation

• Controls:

  • Single stain controls for spectral unmixing

  • Positive interaction controls (known protein pairs)

  • Negative interaction controls (proteins known not to interact)

This methodological framework enables rigorous investigation of FBXL15's interaction with other proteins in cellular contexts, providing spatial information often lost in biochemical approaches.

What are common issues with FBXL15 antibody detection and how can they be resolved?

Researchers working with FBXL15 antibodies may encounter several common challenges. Here are methodological solutions to address them:

How can I distinguish between specific and non-specific signals when studying low-abundance FBXL15 in different cell types?

When studying low-abundance FBXL15 expression, distinguishing genuine signal from background noise requires rigorous methodology:

• Comprehensive controls implementation:

  • Perform parallel experiments with FBXL15 knockdown/knockout cells

  • Include peptide competition controls using the immunizing peptide (FBXL15 fusion protein Ag14949)

  • Use isotype controls to identify non-specific binding of antibody class

• Signal verification with orthogonal techniques:

  • Correlate protein detection with mRNA expression (qRT-PCR)

  • Verify with multiple antibodies targeting different FBXL15 epitopes

  • Combine with FBXL15 overexpression studies in low-expressing cells

• Optimized signal amplification:

  • For Western blot: Extended exposure times, more sensitive ECL substrates

  • For IHC/IF: Tyramide signal amplification, quantum dot conjugates

  • For flow cytometry: Multi-layer detection systems with fluorochrome-labeled tertiary reagents

• Image acquisition optimization:

  • Increase exposure time while monitoring background

  • Implement deconvolution algorithms for improved signal-to-noise ratio

  • Use spectral unmixing to separate autofluorescence from specific signal

• Quantitative analysis approaches:

  • Implement signal intensity thresholding based on negative controls

  • Calculate signal-to-noise ratios across experiments

  • Use statistical approaches to distinguish signal from background variation

• Cell type-specific considerations:

  • Account for autofluorescence characteristics of specific cell types

  • Implement additional blocking steps for cells with high Fc receptor expression

  • Consider fixation modifications for cells with unique membrane compositions

This systematic approach enables confident identification of specific FBXL15 signals even in challenging low-expression contexts.

What advanced bioinformatics approaches can help analyze FBXL15 antibody specificity across different experimental conditions?

Modern bioinformatics tools provide sophisticated approaches for analyzing antibody specificity:

• Epitope prediction and cross-reactivity analysis:

  • Apply biophysics-informed modeling similar to approaches used for antibody specificity prediction

  • Use structural bioinformatics to predict antibody-antigen interactions

  • Implement algorithms that identify potential cross-reactive epitopes across the proteome

• Antibody sequence data mining:

  • Analyze antibody variable regions using methods similar to those applied in database searching approaches

  • Implement sequence similarity scoring for cross-reactivity prediction

  • Apply machine learning algorithms trained on known antibody-antigen interactions

• Experimental data integration:

  • Develop computational workflows integrating proteomics, genomics, and antibody validation data

  • Implement Bayesian networks to estimate probability of specific vs. non-specific binding

  • Create statistical models that incorporate results from multiple validation techniques

• Specificity prediction across conditions:

  • Develop models predicting specificity alterations under different experimental conditions

  • Build condition-specific training datasets from validated antibodies

  • Implement simulation approaches for binding under variable pH, salt, and detergent conditions

• Database-informed validation:

  • Reference FBXL15 expression data from public repositories for validation

  • Cross-reference with proteomics identification databases similar to approaches used for other proteins

  • Implement FBXL15 protein interaction network analysis to validate co-immunoprecipitation results

• Custom algorithms development:

  • Design algorithms specifically for F-box protein antibody validation

  • Build computational tools that analyze antibody validation results across multiple experiments

  • Develop automated decision frameworks for determining antibody reliability

These bioinformatics approaches significantly enhance confidence in antibody specificity assessment while providing insights into potential cross-reactivity under different experimental conditions.

How is FBXL15 antibody being used to elucidate biological roles in disease mechanisms?

FBXL15 antibodies are becoming valuable tools in investigating disease mechanisms:

• Cancer research applications:

  • Used to study FBXL15 expression in human breast cancer tissue

  • Enables investigation of FBXL15's role in cell cycle dysregulation in cancer cells

  • Facilitates identification of potential substrates degraded through FBXL15-mediated ubiquitination in tumors

• Methodological approaches in disease studies:

  • Tissue microarray analysis using optimized IHC protocols (1:50-1:500 dilution)

  • Patient-derived xenograft (PDX) models assessed for FBXL15 expression

  • Correlation of FBXL15 expression with clinical outcomes and treatment response

• Cell-specific expression analysis:

  • Single-cell approaches to identify cell populations with differential FBXL15 expression

  • Spatial transcriptomics combined with IHC to map expression in tissue microenvironments

  • Cell-type specific knockouts to determine tissue-specific functions

• Protein-protein interaction studies:

  • Identification of disease-specific interaction partners through co-immunoprecipitation

  • Proximity labeling approaches to identify spatial interaction networks

  • Comparison of interaction profiles between normal and diseased states

• Post-translational modification mapping:

  • Phospho-specific antibodies to detect disease-associated modifications

  • Ubiquitination site mapping to identify regulatory mechanisms

  • Correlation of modifications with altered FBXL15 function in disease

These approaches collectively build a comprehensive understanding of FBXL15's roles in disease mechanisms, potentially identifying new therapeutic targets.

What novel experimental designs are emerging for studying FBXL15 function in bone development?

Given FBXL15's reported role in bone regulation , several innovative experimental approaches are being developed:

• In vivo models and analyses:

  • Conditional knockout models with bone-specific FBXL15 deletion

  • Temporal regulation using inducible systems to study developmental windows

  • Micro-CT analysis to quantify bone structural parameters

  • Histomorphometric assessment with FBXL15 immunolocalization

• Cell lineage investigations:

  • FBXL15 expression analysis across osteoblast differentiation stages

  • ChIP-seq studies to identify transcriptional targets in bone-forming cells

  • Single-cell RNA-seq combined with FBXL15 protein analysis

  • Lineage tracing of FBXL15-expressing cells during bone development

• Mechanistic studies:

  • Identification of bone-specific FBXL15 substrates via proteomics

  • Analysis of FBXL15 interaction with bone morphogenetic protein (BMP) signaling

  • Investigation of FBXL15's role in osteoblast-osteoclast communication

  • Integration with mechanical loading models to assess mechanosensitive regulation

• Translational approaches:

  • Analysis of FBXL15 variants in patients with bone disorders

  • Correlation of FBXL15 expression with bone mineral density

  • Development of targeted modulation strategies for bone regeneration

  • Ex vivo culture systems to test FBXL15-targeting compounds

• Emerging technologies:

  • Organ-on-chip models incorporating FBXL15 reporter systems

  • CRISPR activation/inhibition screens to identify FBXL15-dependent genes

  • Advanced imaging techniques to visualize FBXL15 dynamics in bone cells in real-time

  • AI-assisted image analysis to quantify FBXL15 expression patterns in bone tissue

These emerging experimental designs will help elucidate FBXL15's specific functions in bone development and homeostasis, potentially leading to novel therapeutic interventions for bone disorders.

How might advances in antibody engineering improve future FBXL15 research tools?

Emerging antibody engineering technologies promise to enhance FBXL15 research capabilities:

• Enhanced specificity engineering:

  • Application of biophysics-informed models to design antibodies with customized specificity profiles

  • Development of antibodies specifically distinguishing between FBXL15 and closely related F-box proteins

  • Creation of conformation-specific antibodies to detect active versus inactive FBXL15 states

• Advanced detection capabilities:

  • Development of split-epitope antibody systems for improved spatial resolution

  • Engineering of antibodies with environmentally-sensitive fluorophores

  • Creation of antibody-based biosensors to monitor FBXL15 activity in real-time

• Novel formats for research applications:

  • Single-domain antibodies (nanobodies) for improved tissue penetration

  • Intrabodies specifically designed for live-cell FBXL15 tracking

  • Bispecific antibodies to simultaneously detect FBXL15 and interaction partners

  • PhotoActivatable antibodies for spatiotemporal control of FBXL15 detection

• Methodological improvements through antibody engineering:

  • Development of recombinant antibody libraries specifically for F-box proteins

  • Generation of antibodies detecting specific post-translational modifications of FBXL15

  • Creation of standardized validation panels for FBXL15 antibody characterization

  • Engineering of antibody-based proximity labeling tools for FBXL15 interaction mapping

• Integration with -omics approaches:

  • Development of antibodies compatible with mass cytometry for high-dimensional analysis

  • Engineering of antibodies for spatial proteomics applications

  • Creation of antibody-DNA conjugates for spatial transcriptomics integration

  • Development of antibodies compatible with database searching in bottom-up proteomics

These advances in antibody engineering will significantly expand the research toolkit available for FBXL15 studies, enabling more precise mechanistic investigations and potential therapeutic applications.