FBXO10 Antibody, HRP conjugated

What is FBXO10 and why is it important in research?

FBXO10 belongs to the F-box protein family, characterized by an approximately 40-amino acid F-box motif. These proteins form part of SCF (SKP1-cullin-F-box) complexes that function as protein-ubiquitin ligases, targeting specific proteins for degradation through the ubiquitin-proteasome pathway. FBXO10 interacts with SKP1 through its F-box domain and with ubiquitination targets through other protein interaction domains . Recent research has revealed FBXO10's significant role in regulating mitochondrial function, including ATP production, membrane potential, and morphological dynamics . Additionally, studies have investigated FBXO10's potential involvement in regulating proteins like BCL2 in the context of B cell lymphoma, though mouse model studies have yielded complex results suggesting possible redundancy with other ubiquitin ligase complexes .

What are the key technical specifications of commonly used FBXO10 antibodies?

FBXO10 antibodies are available in various forms, including polyclonal antibodies like the FBXO10 Rabbit pAb (A14871). This particular antibody has an observed and calculated molecular weight of 105kDa and is recommended for Western blot (1:500-1:2000 dilution) and ELISA applications . The antibody is generated using a recombinant fusion protein containing amino acids 1-180 of human FBXO10 (NP_036298.2) as the immunogen . For storage, these antibodies typically require -20°C conditions with avoidance of freeze/thaw cycles and are usually supplied in PBS buffer with preservatives such as thimerosal and glycerol at specific pH values .

What are the optimal conditions for Western blot analysis using FBXO10 antibodies?

Based on validated protocols, optimal Western blot conditions for FBXO10 detection include:

• Sample preparation: 25μg protein per lane is recommended for tissue lysates such as rat brain .

• Primary antibody dilution: For FBXO10 Rabbit pAb, a 1:1000 dilution provides optimal results, though the working range is 1:500-1:2000 .

• Blocking conditions: 3% nonfat dry milk in TBST has been validated as an effective blocking buffer .

• Secondary antibody: If using an unconjugated primary antibody, HRP Goat Anti-Rabbit IgG at 1:10000 dilution is recommended .

• Detection system: ECL (Enhanced Chemiluminescence) detection systems provide suitable sensitivity, with exposure times around 90 seconds yielding clear results in validated experiments .

• Controls: Include positive control lysates from tissues known to express FBXO10, such as rat brain .

For researchers working with direct HRP-conjugated FBXO10 antibodies, the protocol would need to be adjusted to skip the secondary antibody step while potentially optimizing blocking and washing conditions to minimize background.

How can subcellular localization of FBXO10 be effectively studied?

Research indicates that FBXO10 has distinct subcellular localization patterns, particularly regarding its association with mitochondria. Multiple complementary approaches are recommended for comprehensive analysis:

• Confocal immunofluorescence microscopy: This enables visualization of FBXO10 distribution patterns within cells. Studies have used fluorescent-tagged FBXO10 (such as ptd-Tomato-FBXO10 or GFP-FBXO10) to track localization patterns .

• Subcellular biochemical fractionation: This technique allows separation of mitochondrial fractions from cytosolic components, followed by Western blot analysis using FBXO10 antibodies to quantify distribution patterns .

• Flow cytometry of enriched mitochondrial fractions: This approach enables quantitative assessment of FBXO10 association with isolated intact mitochondria .

Additionally, researchers have employed mutation studies (e.g., FBXO10(C953S) CaaX-mutant) to investigate the mechanisms governing FBXO10's mitochondrial targeting, revealing the importance of geranylgeranylation for proper localization .

What experimental approaches can identify FBXO10 interaction partners and substrates?

To identify FBXO10 interaction partners and potential substrates, several complementary experimental approaches have proven effective:

• Co-immunoprecipitation (Co-IP): Using antibodies against FBXO10 to pull down protein complexes from either whole cell lysates or enriched mitochondrial fractions has successfully identified interactions with proteins such as HSP90, HSP70, and TOM70 .

• Mass spectrometry analysis: Unbiased proteomic analysis of proteins co-purified with affinity-tagged FBXO10 (such as FLAG-FBXO10 or Strep-FBXO10) has helped identify both regulators and potential substrates. For example, this approach identified PGAM5 as a substrate for FBXO10-mediated ubiquitylation and degradation .

• Comparative proteomic analysis: Comparing protein levels in wild-type versus FBXO10-deficient cells (through gene deletion, mutation, or knockdown) helps identify proteins whose stability is regulated by FBXO10-mediated ubiquitylation .

• Functional validation: Confirmation of direct ubiquitylation and degradation requires additional experiments, such as in vitro ubiquitylation assays, cycloheximide chase experiments to measure protein stability, and rescue experiments with proteasome inhibitors .

How can FBXO10 antibodies be used to investigate mitochondrial protein turnover mechanisms?

Recent research has revealed FBXO10's essential role in mitochondrial protein homeostasis, particularly at the outer mitochondrial membrane (OMM). FBXO10 antibodies can be employed in several sophisticated experimental approaches to investigate these processes:

• Differential proteomics: Comparing the mitochondrial proteome between conditions (wild-type, FBXO10-deficient, or expressing mutant variants) can identify proteins whose turnover depends on FBXO10. Research has demonstrated that proteins with significant reciprocal changes in levels (≥2-fold change, 5% FDR) can be identified through mass spectrometry and validated by immunoblotting .

• Ubiquitylation profile analysis: FBXO10 antibodies can be used in conjunction with ubiquitin antibodies to perform sequential immunoprecipitations that isolate and identify ubiquitylated FBXO10 substrates at the OMM. This approach helped identify PGAM5 as an FBXO10 substrate .

• Mitochondrial functional assays: FBXO10 antibodies can complement functional studies measuring mitochondrial ATP production, membrane potential (using probes like TMRM), and morphological dynamics to correlate FBXO10 levels/activity with functional outcomes .

• Spatial organization studies: By combining FBXO10 antibodies with those targeting known mitochondrial markers in super-resolution microscopy, researchers can map the precise spatial organization of FBXO10-containing ubiquitin ligase complexes at the OMM.

What are the key considerations when using FBXO10 antibodies for cross-species research?

When designing experiments involving multiple species, researchers must carefully consider antibody cross-reactivity. The FBXO10 Rabbit pAb (A14871) has documented cross-reactivity with rat FBXO10 , but cross-reactivity with other species may vary and requires validation. Important considerations include:

• Epitope conservation: The immunogen used for antibody generation (amino acids 1-180 of human FBXO10) should be compared across species to predict potential cross-reactivity. Sequence alignment analysis can help identify regions of high or low conservation.

• Validation across species: Positive controls from each species of interest should be included in initial experiments. For the FBXO10 antibody mentioned, rat brain lysate has been validated as a positive control .

• Species-specific modifications: Post-translational modifications may vary across species, potentially affecting antibody recognition. Particular attention should be paid to phosphorylation sites or other modifications within the antibody epitope region.

• Genetic models: Mouse genetic models of FBXO10 (such as the E54K point mutation or frameshift mutation) can provide valuable controls for antibody specificity testing . Western blots comparing wild-type and knockout/mutant tissues help establish antibody specificity.

How can FBXO10 antibodies aid in investigating potential functional redundancy with other F-box proteins?

Studies in mouse models have suggested potential functional redundancy between FBXO10 and other ubiquitin ligase complexes, particularly regarding BCL2 regulation . FBXO10 antibodies can be instrumental in exploring these redundancy mechanisms through several approaches:

• Combinatorial knockdown/knockout studies: Using FBXO10 antibodies alongside antibodies against other F-box proteins in single vs. multiple knockdown/knockout systems can help identify compensatory changes in protein expression or localization.

• Substrate competition assays: In vitro assays using purified components can determine whether multiple F-box proteins can target the same substrate, with FBXO10 antibodies used to immunodeplete specific complexes and assess the impact on substrate ubiquitylation.

• Tissue-specific expression analysis: Immunohistochemistry or immunoblotting with FBXO10 antibodies across multiple tissues can help identify contexts where FBXO10 might be the predominant F-box protein targeting specific substrates versus tissues where redundant mechanisms might operate.

• Developmental studies: Analysis of FBXO10 expression and substrate levels throughout development may reveal temporal windows where redundancy is more or less pronounced, informing the interpretation of genetic model phenotypes.

What are common issues when using FBXO10 antibodies and how can they be resolved?

When working with FBXO10 antibodies, researchers may encounter several technical challenges:

• Background signal: High background in Western blots or immunofluorescence can be addressed by:

  • Optimizing blocking conditions (testing different blocking agents beyond the recommended 3% nonfat dry milk in TBST)

  • Increasing wash duration or frequency

  • Further diluting the primary antibody (testing the range from 1:500 to 1:2000)

  • For HRP-conjugated antibodies specifically, adding reducing agents like 2-mercaptoethanol to the wash buffer may help reduce non-specific disulfide bond formation

• Multiple bands: If observing bands at unexpected molecular weights beyond the expected 105kDa FBXO10 band :

  • Verify sample preparation (complete denaturation, appropriate reducing conditions)

  • Consider potential degradation products or splice variants

  • Include positive and negative controls to help distinguish specific from non-specific bands

• Weak signal: For detection challenges:

  • Reduce antibody dilution (use more concentrated antibody)

  • Increase exposure time (beyond the standard 90 seconds)

  • Enrich for the subcellular fraction where FBXO10 is most abundant (e.g., mitochondrial fractions)

  • Consider using signal amplification systems for low-abundance targets

• Inconsistent results: For reliability issues:

  • Standardize sample preparation (protein concentration, lysis conditions)

  • Avoid antibody freeze/thaw cycles

  • Prepare fresh working dilutions for each experiment

How can researchers validate the specificity of FBXO10 antibody signals in their experimental system?

Validating antibody specificity is crucial for accurate data interpretation. For FBXO10 antibodies, multiple complementary approaches are recommended:

• Genetic controls: Utilize FBXO10 knockout or knockdown systems as negative controls. Mouse models with FBXO10 mutations (E54K point mutation or frameshift mutations) provide excellent validation tools .

• Pre-absorption controls: Pre-incubate the antibody with excess purified FBXO10 antigen (such as the recombinant fusion protein containing amino acids 1-180 of human FBXO10) before application to samples. Specific signals should be eliminated or greatly reduced.

• Multiple antibodies: When possible, confirm key findings using different antibodies targeting distinct epitopes of FBXO10.

• Recombinant expression: Overexpression of tagged FBXO10 (with size-altering tags) should result in an additional band at the expected shifted molecular weight.

• Subcellular fractionation: Given FBXO10's known mitochondrial association , enrichment of signal in mitochondrial fractions provides supporting evidence for specificity.

What are the key considerations when interpreting FBXO10 expression data across different cell types and tissues?

When analyzing FBXO10 expression patterns, several factors require careful consideration:

• Tissue-specific expression: FBXO10 expression varies across tissues, with implications for interpreting comparative studies. The search results indicate FBXO10 is detectable in rat brain , but expression levels in other tissues and species require validation.

• Subcellular distribution patterns: FBXO10's distinct localization to mitochondria is functionally significant . Interpretation of total FBXO10 levels should consider potential redistribution between subcellular compartments, which may occur without changes in total protein expression.

• Context-dependent interactions: FBXO10's association with proteins like HSP90, HSP70, and TOM70 may vary across cell types, influencing both antibody accessibility and FBXO10 function. These interaction partners should be considered when comparing FBXO10 detection across different cellular contexts.

• Post-translational modifications: FBXO10 undergoes geranylgeranylation that affects its localization and function . Antibodies may have differential recognition of modified versus unmodified forms, potentially leading to misleading results if not properly controlled.

• Splice variants: Consider the potential for alternative splice forms when interpreting unexpected banding patterns, though current evidence suggests limited splice variation for FBXO10 .

• Experimental manipulations: Interventions like HSP90 inhibition with CCT018159 can affect FBXO10 localization , potentially influencing antibody detection efficiency in certain subcellular compartments.

How might FBXO10 antibodies contribute to understanding mitochondrial dysfunction in disease models?

FBXO10's role in regulating mitochondrial protein turnover, ATP production, and membrane potential suggests its potential involvement in mitochondrial pathologies. FBXO10 antibodies could be instrumental in several emerging research areas:

• Neurodegenerative disorders: Given FBXO10's expression in brain tissue and its role in mitochondrial homeostasis , antibodies could help investigate potential dysregulation of FBXO10-dependent protein turnover in conditions like Alzheimer's and Parkinson's diseases where mitochondrial dysfunction is implicated.

• Cancer metabolism: Studies could employ FBXO10 antibodies to compare expression and localization patterns between normal and cancer cells, particularly focusing on cancers with altered mitochondrial metabolism or resistance to apoptosis.

• Aging research: Age-related changes in FBXO10 expression, localization, or substrate specificity could be monitored using appropriate antibodies, potentially linking impaired mitochondrial protein quality control to aging phenotypes.

• Drug screening: FBXO10 antibodies could facilitate high-throughput screening of compounds that modulate FBXO10 expression, localization, or activity as potential therapeutic interventions for mitochondrial disorders.

What technical advances might enhance the utility of FBXO10 antibodies in complex experimental systems?

Several technological developments could expand the applications of FBXO10 antibodies in sophisticated research contexts:

• Proximity labeling approaches: Combining FBXO10 antibodies with proximity labeling technologies (BioID, APEX) could help identify transient or context-specific interaction partners and substrates in intact cellular environments.

• Single-cell analysis: Adapting FBXO10 antibodies for single-cell proteomic techniques would allow researchers to investigate cell-to-cell variation in FBXO10 expression and function within heterogeneous tissues or populations.

• Live-cell imaging compatible antibodies: Development of membrane-permeable antibody fragments or nanobodies against FBXO10 could enable real-time monitoring of FBXO10 dynamics in living cells.

• Multiplexed detection systems: Creation of FBXO10 antibodies compatible with multiplexed imaging techniques (Imaging Mass Cytometry, CODEX) would facilitate simultaneous analysis of FBXO10 alongside numerous other proteins in tissue sections.

• Substrate-specific antibodies: Development of antibodies that specifically recognize FBXO10-substrate complexes or ubiquitylated forms of known FBXO10 substrates would enhance studies of FBXO10's enzymatic activity in various contexts.

How can researchers leverage FBXO10 antibodies to explore the therapeutic potential of targeting FBXO10-dependent pathways?

FBXO10's involvement in protein degradation pathways presents potential therapeutic opportunities that could be explored using appropriate antibodies:

• Biomarker development: FBXO10 antibodies could help evaluate whether FBXO10 expression, localization, or activity correlates with disease progression or treatment response, potentially serving as diagnostic or prognostic biomarkers.

• Target validation: In disease models where FBXO10 dysfunction is implicated, antibodies can help validate FBXO10 or its substrates as therapeutic targets by monitoring expression changes and downstream effects following experimental interventions.

• Drug mechanism studies: For compounds designed to modulate FBXO10 activity or its interaction with specific substrates, antibodies provide essential tools for confirming mechanism of action and target engagement in cellular and animal models.

• Combination therapy assessment: When studying treatments targeting multiple components of ubiquitin-proteasome pathways, FBXO10 antibodies can help assess pathway compensation or synergistic effects that might inform optimal therapeutic combinations.

• Patient stratification: In personalized medicine approaches, analysis of FBXO10 expression or activity patterns using specific antibodies might help identify patient subgroups most likely to benefit from targeted therapies affecting FBXO10-dependent degradation pathways.