FBXO3 Antibody, Biotin conjugated

What is FBXO3 and why is it a significant research target?

FBXO3 is a ubiquitin E3 ligase F box component that potently stimulates cytokine secretion from human inflammatory cells by mediating the degradation of the TRAF inhibitory protein, Fbxl2 . Its significance as a research target stems from its role in innate immunity, as it conveys signals from cell surface receptors to elicit transcriptional activation of genes encoding pro-inflammatory cytokines . The bacterial-like ApaG molecular signature in its carboxyl-terminal structure is indispensable for mediating Fbxl2 disposal and stimulating cytokine secretion . Targeting FBXO3 pharmacologically might represent a promising strategy for immune-related disorders characterized by heightened host inflammatory responses, as demonstrated in murine models of viral pneumonia, septic shock, colitis, and cytokine-driven systemic inflammation .

What are the optimal applications for biotin-conjugated FBXO3 antibodies?

Biotin-conjugated FBXO3 antibodies are particularly valuable for immunodetection techniques that leverage the strong biotin-streptavidin interaction. While specific applications for biotin-conjugated variants aren't explicitly detailed in the search results, FBXO3 antibodies are generally suitable for Western Blotting (WB), ELISA, Flow Cytometry (FACS), and Immunohistochemistry (IHC) . For Western Blotting applications, a recommended dilution ratio of 1:500-1:2000 has been established . The biotin conjugation enables signal amplification through secondary detection with streptavidin-coupled enzymes or fluorophores, making these antibodies particularly useful for detecting low-abundance FBXO3 proteins in complex biological samples or tissue sections.

What is the recommended storage protocol for maintaining antibody activity?

The optimal storage conditions for FBXO3 antibodies include maintaining them in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3, kept at -20°C for up to 12 months . Researchers should diligently avoid repeated freeze/thaw cycles as these can progressively degrade antibody integrity and performance . While most antibodies are best stored at -20°C, directly-labeled flow cytometry antibodies require storage at 2-8°C . Although the guaranteed shelf life is typically one year, antibodies often remain functional beyond this period, but performance should be validated before use in critical experiments . For short-term storage after dilution or initial use, refrigeration at 4°C can preserve antibody activity for approximately one week, though performance may gradually diminish with each reuse .

How is antibody specificity validated for FBXO3 detection?

Validation of FBXO3 antibody specificity involves multiple complementary approaches. Western blotting serves as a primary validation method, where the antibody should detect a protein band at the expected molecular weight of 48-50 kDa for FBXO3 . Additional validation may include immunoprecipitation followed by mass spectrometry analysis to confirm target identity. For immunostaining applications, specificity can be verified using knockout/knockdown controls or peptide competition assays where pre-incubation with the immunizing peptide should abolish specific staining. The search results indicate that some FBXO3 antibodies are generated from rabbits immunized with KLH-conjugated synthetic peptides corresponding to specific amino acid sequences (e.g., 395-422) from the C-terminal region of human FBXO3 , providing epitope-specific recognition. Validation across multiple cell lines or tissues with known differential expression of FBXO3 can further confirm antibody specificity and reliability.

What sample preparation techniques ensure optimal FBXO3 detection?

For optimal FBXO3 detection, sample preparation techniques should preserve protein integrity while maximizing accessibility of the target epitope. For Western blotting, samples should be lysed in buffers containing appropriate protease inhibitors to prevent degradation of FBXO3 (48-50 kDa) . When performing immunohistochemistry, appropriate antigen retrieval methods are essential - common approaches include Tris-EDTA Buffer (pH 9.0) or Citrate Buffer (pH 6.0) with heat-induced retrieval either via water bath heating (15 minutes at boiling temperature followed by natural cooling) or microwave retrieval (5 minutes at high power, 3 minutes off, then 5 minutes at medium power followed by natural cooling) . For flow cytometry, gentle cell fixation and permeabilization protocols that preserve epitope integrity are recommended. Since FBXO3 functions within the ubiquitin-proteasome system, researchers may consider adding deubiquitinase inhibitors to lysates when studying FBXO3's interactions with target proteins in the ubiquitination pathway.

How can biotin-conjugated FBXO3 antibodies be utilized in multi-parameter flow cytometry?

In multi-parameter flow cytometry, biotin-conjugated FBXO3 antibodies offer distinct advantages due to their flexibility in detection strategies. Researchers should implement a sequential staining protocol beginning with surface markers followed by fixation, permeabilization, and intracellular FBXO3 staining. The biotin conjugate can be detected using streptavidin coupled to fluorophores with emission spectra distinct from other fluorochromes in the panel, effectively minimizing spectral overlap. When designing panels, consider using streptavidin-coupled fluorophores like BV421, BV650, or APC-Cy7 depending on the cytometer configuration and other fluorophores employed. Titration experiments are essential to determine optimal antibody concentration that maximizes signal-to-noise ratio. For co-localization studies investigating FBXO3's interaction with other proteins in the ubiquitination pathway such as Fbxl2 or TRAF proteins , the biotin-streptavidin system provides signal amplification beneficial for detecting subtle changes in expression levels following experimental treatments. Compensation controls must include cells stained only with the biotin-FBXO3 antibody and streptavidin-fluorophore to account for any spillover into other detection channels.

What experimental approaches can elucidate FBXO3's role in ubiquitination pathways?

To investigate FBXO3's function in ubiquitination pathways, researchers should implement a multi-faceted experimental approach. In vitro ubiquitination assays can be constructed similar to those described in the literature, utilizing 50 mM Tris pH 7.6, 5 mM MgCl₂, 0.6 mM DTT, 2 mM ATP, 1.5 ng/μl E1, 10 ng/μl Ubc5, 10 ng/μl Ubc7, 1 μg/μl ubiquitin, 1 μM ubiquitin aldehyde, and purified Cullin1, Skp1, Rbx1, along with in vitro synthesized FBXO3 . Co-immunoprecipitation experiments using biotin-conjugated FBXO3 antibodies can identify novel protein interactions within the ubiquitination machinery, with subsequent mass spectrometry analysis to characterize the complete interactome. Proximity ligation assays (PLA) offer powerful visualization of protein-protein interactions between FBXO3 and potential substrates in situ. For substrate identification, global proteomics comparing protein expression and ubiquitination patterns in FBXO3 knockout/knockdown versus control cells can reveal putative targets. Additionally, researchers should consider utilizing BC-1215 or other FBXO3 inhibitors at concentrations ranging from 10⁻¹¹ to 10⁻⁴ M to disrupt FBXO3-substrate interactions and observe resultant changes in ubiquitination patterns .

How can structure-function studies of FBXO3 be designed using domain-specific antibodies?

Structure-function studies of FBXO3 can be strategically designed using domain-specific antibodies that target discrete regions of the protein. Researchers should procure or develop antibodies targeting different domains, including the F-box domain (required for SCF complex interaction) and the ApaG domain (essential for substrate recognition) . Immunoprecipitation experiments comparing antibodies targeting N-terminal versus C-terminal epitopes can reveal domain-specific protein interactions. Domain deletion mutants (such as FBXO3-C278 lacking the ApaG domain or FBXO3-N70 lacking the N-terminal F-box domain) can be expressed in cellular systems to evaluate functional consequences . These mutants can then be immunoprecipitated using domain-specific antibodies to assess how domain deletion affects interaction partners. For advanced structural insights, researchers should consider using domain-specific antibodies in hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map conformational changes upon substrate binding. Additionally, native protein immunoprecipitation followed by limited proteolysis can elucidate domain accessibility and structural arrangements. Comparing results from antibodies targeting different epitopes within the ApaG domain (amino acids 395-422) can provide granular insights into the structural requirements for Fbxl2 recognition .

What are the critical controls for validating FBXO3 antibody specificity in co-localization studies?

For robust validation of FBXO3 antibody specificity in co-localization studies, a comprehensive set of controls is essential. Primary negative controls should include FBXO3 knockout or knockdown samples, which should show substantially reduced or absent signal compared to wild-type samples. Peptide competition assays where the biotin-conjugated antibody is pre-incubated with excess immunizing peptide (from the 395-422 amino acid region) should abolish specific staining while non-specific binding may persist. Isotype controls using biotin-conjugated IgG of the same isotype and host species (rabbit IgG in this case) are necessary to distinguish specific staining from Fc receptor-mediated binding. For positive controls, researchers should overexpress tagged FBXO3 and confirm co-localization between the biotin-conjugated antibody signal and the tag-specific antibody signal. When studying co-localization with interaction partners like Fbxl2, sequential staining protocols must be optimized to prevent cross-reactivity, and single-stained samples are required to establish proper thresholds for co-localization analysis. Additionally, secondary reagent-only controls (streptavidin-fluorophore without primary antibody) should be included to account for non-specific streptavidin binding to endogenous biotin in the sample.

How can biotin-conjugated FBXO3 antibodies be employed in studying inflammation mechanisms?

Biotin-conjugated FBXO3 antibodies offer versatile applications for investigating inflammation mechanisms, particularly given FBXO3's role in cytokine-driven inflammatory responses . Researchers should design multi-parameter flow cytometry panels that simultaneously detect FBXO3 and inflammatory markers in immune cells following stimulation with lipopolysaccharide (LPS) or other inflammatory triggers. For mechanistic studies, time-course experiments tracking FBXO3 expression and localization during inflammatory activation can reveal dynamic regulation patterns. Chromatin immunoprecipitation (ChIP) assays utilizing biotin-conjugated FBXO3 antibodies can identify potential direct interactions with chromatin or transcription factors regulating inflammatory genes. In tissue sections from inflammatory disease models, dual immunofluorescence staining can map FBXO3 expression in relation to infiltrating immune cells and inflammatory markers. For functional studies, researchers should compare the effects of FBXO3 inhibitors (like the benzathine-based derivatives) on cytokine production in human blood mononuclear cells , using biotin-conjugated FBXO3 antibodies to track changes in protein expression and localization. Additionally, proximity ligation assays can visualize interactions between FBXO3 and TRAF proteins in situ, providing spatial context to biochemical findings on FBXO3's role in regulating TRAF stability through the modulation of Fbxl2 .

What are the optimal dilution ratios for different experimental applications?

Each application requires careful optimization, with preliminary titration experiments recommended to determine the ideal dilution for specific experimental conditions. The biotin conjugation may necessitate slightly different dilution ratios compared to unconjugated antibodies due to potential differences in binding efficiency and signal amplification through the biotin-streptavidin interaction.

How does FBXO3 expression vary across tissue and cell types?

Based on available research data, FBXO3 expression patterns demonstrate notable tissue and cell-type specificity that researchers should consider when designing experiments:

This expression profile suggests that researchers should select appropriate cellular models when studying FBXO3 function, with particular emphasis on immune and inflammatory cell types where FBXO3 appears to play significant roles. Expression levels may change dramatically under inflammatory conditions, necessitating careful experimental timing when studying FBXO3 regulation.

What troubleshooting strategies address common challenges with biotin-conjugated antibodies?

When experiencing detection difficulties specifically with the ApaG domain of FBXO3, researchers should consider that this domain's structural features (containing several beta-sheets) may affect epitope accessibility in certain applications, potentially requiring adjustments to sample preparation protocols.

How can biotin-conjugated FBXO3 antibodies advance drug development research?

Biotin-conjugated FBXO3 antibodies represent valuable tools for advancing drug development research, particularly for compounds targeting the inflammation pathway. The development of FBXO3 inhibitors, such as the benzathine-based derivatives that target the ApaG motif, has demonstrated potential therapeutic applications in reducing cytokine-driven inflammation . Researchers can employ these antibodies in high-throughput screening assays to identify novel compounds that alter FBXO3 expression, localization, or function. Protein interaction assays utilizing biotin-conjugated antibodies can evaluate how candidate drugs affect FBXO3's binding to Fbxl2 at concentrations ranging from 10⁻¹¹ to 10⁻⁴ M . For mechanistic studies, these antibodies enable visualization of FBXO3 localization changes in response to drug treatment through immunofluorescence microscopy. Target engagement assays can be designed where drug-induced conformational changes in FBXO3 alter antibody binding profiles, providing direct evidence of compound-target interaction. The biotin conjugation facilitates multiplex assays that simultaneously evaluate drug effects on FBXO3 and inflammatory markers, offering comprehensive insights into compound efficacy. Additionally, these antibodies can support pharmacodynamic biomarker development by quantifying FBXO3 levels in clinical samples from patients receiving experimental therapeutics targeting the inflammatory pathway.

How does FBXO3 interact with the broader ubiquitin-proteasome system?

FBXO3 functions as a substrate recognition component within the SCF (SKP1-CUL1-F-box protein) E3 ubiquitin ligase complex , representing a critical node in the broader ubiquitin-proteasome system with multiple interaction pathways:

This complex network positions FBXO3 as a multifunctional regulator within the ubiquitin-proteasome system, affecting both direct substrates and downstream signaling pathways. Researchers should consider these interactions when designing experiments to study FBXO3 function, particularly when interpreting results from inhibition or knockdown studies where multiple pathways may be affected.