FBXO40 Antibody

What is FBXO40 and what cellular functions does it regulate?

FBXO40 functions as a substrate-recognition component of the SCF (SKP1-CUL1-F-box protein) complex, a type of E3 ubiquitin ligase involved in protein degradation pathways. The protein plays a crucial role in targeting specific proteins for degradation through the ubiquitin-proteasome system and appears to function specifically in myogenesis (muscle development) . Dysregulation of FBXO40 has been implicated in various diseases, including cancer and neurodegenerative disorders, making it an important research target for understanding disease mechanisms .

What types of FBXO40 antibodies are currently available for research?

Several types of FBXO40 antibodies are available for research applications, with variations in host species, clonality, and target epitopes:

Most commercially available antibodies target internal epitopes of the FBXO40 protein and are available in unconjugated formulations .

Which applications are FBXO40 antibodies validated for, and what are the recommended dilutions?

FBXO40 antibodies have been validated for several research applications, with specific recommended dilution ranges:

For optimal results, researchers should perform antibody titration experiments to determine the ideal concentration for their specific experimental conditions and sample types .

How should I design my experimental controls when using FBXO40 antibodies?

When designing experiments with FBXO40 antibodies, incorporate these essential controls:

• Positive control tissues/cells: Human skeletal muscle tissue has been validated for FBXO40 expression and can serve as a positive control for antibody validation .

• Peptide competition assay: A 150kDa band observed in Western blots has been successfully blocked by incubation with the immunizing peptide, confirming specificity despite the discrepancy with the predicted molecular weight .

• Loading controls: In Western blot applications, include appropriate loading controls such as GAPDH, as used in previous studies .

• Secondary antibody controls: Include secondary-only controls to assess potential non-specific binding, using infrared-fluorescent conjugated secondary antibodies like IRDye 800CW donkey anti-goat IgG for goat primary antibodies or Alexa Fluor 680 rabbit anti-mouse IgG for mouse primary antibodies .

Why does the FBXO40 protein show a 150kDa band in Western blot when its calculated molecular weight is 79.8kDa?

This molecular weight discrepancy represents an unresolved question in the field. Preliminary experiments with FBXO40 antibodies have consistently detected an approximately 150kDa band in human colon, duodenum, ileum, heart, and uterus lysates, despite the calculated size of 79.8kDa according to the reference sequence NP_057382.2 .

Several possible explanations exist for this discrepancy:

• Post-translational modifications: FBXO40 may undergo extensive modifications such as glycosylation, phosphorylation, or ubiquitination that significantly increase its apparent molecular weight.

• Alternative splicing: Uncharacterized splice variants may exist that are larger than the canonical form.

• Protein complexes: FBXO40 might remain tightly bound to other proteins even under denaturing conditions.

The specificity of this 150kDa band has been confirmed through peptide blocking experiments, where incubation with the immunizing peptide successfully abolished the signal . Researchers are encouraged to report their findings with different antibodies or lysates to help resolve this ongoing question in the field.

How can I optimize blocking conditions for Western blot detection of FBXO40?

Based on published protocols, two effective blocking approaches have been documented:

• BSA-based blocking: Block membranes with 5% BSA in PBS for 1 hour at room temperature before incubating with FBXO40 primary antibody (e.g., FBXO40 antibody from Abnova) diluted 1:200 in 5% BSA in PBS at 4°C overnight .

• Odyssey Blocking Buffer: For infrared detection systems, incubate secondary antibodies diluted 1:5000 in a solution containing 50% Odyssey® Blocking Buffer with 0.01% SDS in PBS or TBST for enhanced signal-to-noise ratio .

Optimization experiments comparing different blocking reagents (BSA, milk, commercial blocking buffers) may be necessary to determine the best conditions for your specific antibody and detection system.

How can FBXO40 antibodies be used to investigate protein degradation pathways in muscle development and disease?

FBXO40 antibodies provide valuable tools for investigating protein degradation pathways, particularly in muscle development and muscle-related diseases:

• Myogenesis research: FBXO40 functions in myogenesis, making it relevant for studying muscle development. Researchers can use these antibodies to track FBXO40 expression patterns during different stages of myoblast differentiation and muscle regeneration .

• Relationship with other regulatory proteins: FBXO40 can be studied alongside other factors involved in muscle regulation. Previous research has examined FBXO40 in conjunction with MURF1, FOXO1, FOXO3, EIF3F, MYOD, and myogenin—all key regulators of muscle development and atrophy .

• Aging and muscle atrophy: Studies have investigated the regulation of the ubiquitin-proteasome system in aging models, with FBXO40 antibodies being used alongside antibodies against other components of protein degradation pathways .

For these advanced applications, researchers should consider dual-labeling approaches and carefully validated antibody combinations to investigate protein interactions and regulatory networks.

What are the considerations for using FBXO40 antibodies in studies of cancer and neurodegenerative disorders?

When investigating the role of FBXO40 in disease pathogenesis:

• Tissue-specific expression analysis: FBXO40 has shown expression in multiple human tissues including colon, duodenum, ileum, heart, uterus, and skeletal muscle . When studying disease models, consider the tissue-specific context and potential variations in expression.

• Comparison with normal tissues: Include appropriate normal tissue controls when examining disease samples, as baseline expression may vary across tissues and cell types.

• Integration with other SCF complex components: Consider analyzing other components of the SCF complex alongside FBXO40 to obtain a more comprehensive understanding of E3 ligase function in disease contexts.

• Target protein identification: Design experiments to identify the specific proteins targeted by FBXO40 for ubiquitination in different disease contexts, which may require co-immunoprecipitation approaches followed by mass spectrometry.

What are the optimal storage conditions for FBXO40 antibodies?

To maintain antibody performance and stability:

• Store FBXO40 antibodies at -20°C upon receipt to preserve functionality .

• Minimize freeze-thaw cycles, as repeated freezing and thawing can degrade antibody quality and reduce binding efficiency .

• Consider preparing working aliquots of the antibody to avoid repeated freeze-thaw cycles of the entire stock.

• For short-term use (within 1-2 weeks), antibodies can be stored at 4°C in their appropriate buffer systems containing preservatives such as 0.02% sodium azide .

How should I prepare working solutions for different experimental applications?

For optimal results in different applications:

• ELISA applications: Dilute antibodies according to their specified ranges (e.g., 1:2000-1:10000) in appropriate blocking buffer. The detection limit for peptide ELISA has been reported as 1:128000 dilution for some FBXO40 antibodies .

• Western blot applications: Prepare antibody dilutions in blocking buffer (e.g., 5% BSA in PBS) with reported concentrations around 0.1μg/ml being effective for detecting the approximately 150kDa band in human tissue lysates .

• Immunohistochemistry: Prepare working dilutions between 1:20-1:200 as recommended for paraffin-embedded tissue sections .

When preparing working solutions, maintain sterile conditions and include preservatives for solutions that will be stored for extended periods. Filter-sterilized buffers containing 0.02% sodium azide are commonly used for this purpose .

How should I validate the specificity of FBXO40 antibodies for my experimental system?

To ensure antibody specificity and reliable results:

• Peptide competition assays: Pre-incubate the antibody with its immunizing peptide (e.g., peptide with sequence C-KEPQENQKQQDVRT for some goat polyclonal antibodies) before application to your samples. The disappearance of the specific band or staining pattern confirms antibody specificity .

• Multiple antibody validation: When possible, compare results using antibodies from different sources or those targeting different epitopes of FBXO40.

• Known positive tissues: Include human skeletal muscle, colon, duodenum, ileum, heart, or uterus samples as positive controls, as FBXO40 expression has been documented in these tissues .

• Genetic approaches: For definitive validation, consider using FBXO40 knockdown/knockout systems where available to confirm antibody specificity.

What considerations should I keep in mind when interpreting FBXO40 expression patterns in different experimental contexts?

When analyzing FBXO40 expression:

• Molecular weight considerations: Be aware of the discrepancy between the calculated molecular weight (79.8kDa) and the observed band at approximately 150kDa in Western blot applications. This discrepancy is consistently reported across multiple antibodies and tissue types but remains unexplained in the literature .

• Expression in different tissues: FBXO40 has been detected in various human tissues. Consider tissue-specific expression patterns when designing experiments and interpreting results .

• Relationship to muscle biology: Given FBXO40's role in myogenesis, expression patterns may correlate with muscle development stages or pathological conditions affecting muscle tissue .

• Integration with pathway analysis: Interpret FBXO40 expression in the context of the ubiquitin-proteasome pathway and related regulatory networks, including other F-box proteins and E3 ligase complex components.