APC10 Antibody

What is APC10 and why is it important in cellular research?

APC10 (also known as ANAPC10 or DOC1) is a highly conserved component of the anaphase-promoting complex/cyclosome (APC/C), which functions as a cell cycle-regulated E3 ubiquitin ligase. This complex plays a crucial role in controlling progression through mitosis and the G1 phase of the cell cycle. APC/C is responsible for degrading anaphase inhibitors, mitotic cyclins, and spindle-associated proteins, ensuring that mitotic events occur in the proper sequence. APC10 specifically serves as a substrate recognition subunit that regulates the binding of specific substrates to the APC/C complex, similar to the function of coactivators . Recent research has also revealed that APC10 interacts with NLRP3 to modulate inflammasome activation during different cell cycle phases, establishing a connection between cell division and innate immune responses . Given these critical functions, APC10 antibodies are valuable tools for investigating cell cycle regulation, protein degradation pathways, and inflammatory processes.

What types of APC10 antibodies are available for research applications?

Several types of APC10 antibodies are available for research applications, varying in host species, clonality, and epitope targets:

• Polyclonal antibodies: These antibodies, such as the rabbit polyclonal anti-APC10 antibody, recognize multiple epitopes of the APC10 protein. For example, Novus Biologicals offers a rabbit polyclonal antibody raised against a 16 amino acid synthetic peptide near the center of human APC10 (within amino acids 60-110) .

• Monoclonal antibodies: These provide higher specificity for single epitopes, such as the mouse monoclonal antibody clone 3E9A9 available from Invitrogen .

Researchers should select antibodies based on their specific application needs and the species being studied. Most APC10 antibodies show reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across these species . Both N-terminal and C-terminal targeting antibodies are available, which can be particularly useful when studying potential truncated variants or confirming full-length protein detection .

What are the validated applications for APC10 antibodies?

APC10 antibodies have been validated for multiple experimental applications, with varying degrees of optimization:

• Western Blotting: Most commercial APC10 antibodies are validated for western blot applications, allowing detection of full-length APC10 protein and potential truncated variants .

• Immunoprecipitation (IP): Both N-terminal and C-terminal APC10 antibodies have been successfully used for immunoprecipitation studies, helping researchers identify protein-protein interactions involving APC10 .

• Immunohistochemistry (IHC): Several antibodies are validated for both paraffin-embedded and frozen tissue sections .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): Antibodies such as the rabbit polyclonal from Novus Biologicals have been validated for cellular localization studies through immunofluorescence techniques .

• ELISA: Some antibodies, including both monoclonal and polyclonal options, are suitable for enzyme-linked immunosorbent assays .

When selecting an antibody for a specific application, researchers should review validation data provided by manufacturers and consider published studies that have successfully employed these antibodies in similar experimental systems.

What are the optimal storage and handling conditions for APC10 antibodies?

Proper storage and handling of APC10 antibodies are critical for maintaining their activity and specificity. Based on manufacturer recommendations for commercial APC10 antibodies:

Following these guidelines will help preserve antibody function and ensure consistent experimental results across multiple studies.

How can I validate the specificity of APC10 antibodies in my experimental system?

Validating antibody specificity is crucial for generating reliable results, especially given reports of cross-reactivity with non-target proteins. A systematic validation approach should include:

• RNAi knockdown validation: Reduce APC10 expression through siRNA or shRNA and confirm corresponding reduction in antibody signal. This approach has been successfully used to validate several APC antibodies .

• Use of multiple antibodies: Compare results using antibodies targeting different epitopes (N-terminal vs. C-terminal) of APC10. Consistent detection patterns across different antibodies increase confidence in specificity .

• Immunoprecipitation followed by mass spectrometry: This can definitively identify proteins recognized by the antibody.

• Western blot analysis in cell lines with known APC10 expression profiles: Include positive controls (cells with high APC10 expression) and negative controls when possible.

• Be cautious of non-specific bands: Several APC10 antibodies have been reported to detect a consistent 150 kDa protein that is unlikely to be APC10. This band appears with both N-terminal and C-terminal antibodies . When performing western blot analysis, carefully evaluate all detected bands and do not assume all recognized proteins are APC10 or its variants.

• Blocking peptide competition: Pre-incubation of the antibody with the immunizing peptide should abolish specific signals in your assay.

How can APC10 antibodies be used to investigate APC10's role in NLRP3 inflammasome regulation?

Recent research has revealed that APC10 plays a critical role in modulating NLRP3 inflammasome activation during different cell cycle phases . To investigate this relationship using APC10 antibodies:

• Co-immunoprecipitation studies: Use APC10 antibodies to pull down protein complexes and probe for NLRP3 association. This can help determine if and when these proteins interact during different cell cycle phases.

• Cell cycle synchronization experiments: Synchronize cells at different stages of the cell cycle (interphase vs. mitosis) and use immunofluorescence with APC10 and NLRP3 antibodies to examine their co-localization patterns. During interphase, APC10 interacts with NLRP3 to promote inflammasome activation, whereas during mitosis, this interaction is disrupted .

• Proximity ligation assays (PLA): This technique can visualize and quantify protein-protein interactions between APC10 and NLRP3 with greater sensitivity than traditional co-immunofluorescence.

• Dual immunostaining in inflammatory disease models: Examine the relationship between cell cycle progression and inflammasome activation in tissues from inflammatory disease models using APC10 and NLRP3 antibodies.

• Live-cell imaging: Using fluorescently tagged antibody fragments or nanobodies against APC10 and NLRP3 allows visualization of their dynamic interactions during cell cycle progression.

These approaches can provide valuable insights into how cell cycle regulation interfaces with inflammatory responses, potentially revealing new therapeutic targets for inflammatory disorders.

What are the best practices for using APC10 antibodies in co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is valuable for studying APC10's interactions with other proteins. Evidence shows that APC10 physically interacts with other APC/C components and also forms complexes with proteins like NLRP3 . For optimal Co-IP results:

• Antibody selection: Choose antibodies validated for immunoprecipitation applications. Both N-terminal and C-terminal antibodies have successfully pulled down APC10 and its binding partners in previous studies .

• Cell lysis conditions: Use gentle lysis buffers (typically containing 0.5-1% NP-40 or Triton X-100) to preserve protein-protein interactions. The composition may need adjustment depending on the interaction strength between APC10 and your protein of interest.

• Pre-clearing lysates: To reduce non-specific binding, pre-clear cell lysates with appropriate control IgG and protein A/G beads before adding the specific antibody.

• Controls: Always include an isotype-matched control antibody (such as normal rabbit IgG for rabbit polyclonal APC10 antibodies) to identify non-specific binding . For reverse Co-IP validation, immunoprecipitate with antibodies against the suspected interacting partner and blot for APC10.

• Confirmation strategy: Follow IP with western blotting using antibodies recognizing different epitopes than those used for immunoprecipitation. Previous studies have confirmed APC10 interactions by using N-terminal antibodies for IP and C-terminal antibodies for detection (or vice versa) .

• Crosslinking consideration: For transient or weak interactions, consider mild chemical crosslinking before cell lysis.

These methodological considerations will help ensure that detected interactions are specific and physiologically relevant.

How can I distinguish between full-length APC10 and potential truncated variants in western blot analysis?

Distinguishing between full-length APC10 and potential truncated variants requires careful experimental design and antibody selection:

• Use antibodies targeting different regions: Employ both N-terminal and C-terminal antibodies in parallel experiments. Full-length APC10 will be detected by both, while N-terminal fragments will only be detected by N-terminal antibodies, and C-terminal fragments only by C-terminal antibodies .

• Size reference: Full-length human APC10 has a molecular weight of approximately 21-24 kDa . Any bands substantially smaller may represent truncated variants or degradation products.

• Positive controls: Include lysates from cell lines known to express full-length APC10, such as HCT116 cells, which have been confirmed to express the intact protein .

• Negative controls or comparisons: Consider using lysates from cell lines with known APC10 truncations, if available. For example, SW480 cells express a truncated form of APC that can serve as a reference point .

• Be wary of the 150 kDa band: Multiple APC10 antibodies detect a consistent 150 kDa protein that is unlikely to be APC10. This band appears with both N-terminal and C-terminal antibodies and should not be mistaken for APC10 .

• Validate with knockdown experiments: siRNA-mediated knockdown of APC10 should reduce the intensity of bands representing authentic APC10 protein, while non-specific bands will remain unchanged.

These strategies will help researchers accurately identify and characterize APC10 variants in their experimental systems.

Why do I observe a 150 kDa band when using APC10 antibodies in Western blotting?

The consistent detection of a 150 kDa protein by various APC10 antibodies has been widely reported and presents a potentially confusing issue for researchers . To understand and address this phenomenon:

Understanding this consistent cross-reactivity helps researchers avoid misinterpretation of western blot results and ensures accurate identification of authentic APC10 protein.

What controls should I include when using APC10 antibodies in immunofluorescence studies?

Proper controls are essential for generating reliable immunofluorescence data with APC10 antibodies:

• Primary antibody controls:

  • Isotype control: Use an isotype-matched irrelevant antibody at the same concentration as the APC10 antibody to assess non-specific binding.

  • Absorption control: Pre-incubate the APC10 antibody with excess immunizing peptide (if available) to block specific binding sites.

• Secondary antibody control: Omit the primary antibody but include the secondary antibody to detect non-specific secondary antibody binding.

• Biological controls:

  • Positive control: Include cells or tissues known to express APC10, such as actively dividing cells where APC/C is functionally important.

  • Negative control: When possible, include samples with reduced or absent APC10 expression (e.g., APC10 knockdown cells).

• Cell cycle markers: Since APC10 function varies across the cell cycle, co-stain with cell cycle phase markers (e.g., phospho-histone H3 for mitosis) to correlate APC10 localization with cell cycle stages.

• Subcellular marker controls: Co-stain with markers for specific subcellular compartments (e.g., DAPI for nucleus, mitochondrial markers) to accurately determine APC10 localization.

• Technical controls:

  • Autofluorescence control: Examine unstained samples to identify any intrinsic fluorescence.

  • Cross-channel bleed-through control: In multi-color experiments, include single-stained controls to assess signal overlap between channels.

Implementing these controls will help ensure that observed signals truly represent APC10 localization rather than artifacts or non-specific binding.

How can I optimize immunohistochemistry protocols for APC10 detection in tissue samples?

Optimizing immunohistochemistry (IHC) protocols for APC10 detection requires careful consideration of several parameters:

• Fixation and antigen retrieval:

  • Start with standard formalin fixation and paraffin embedding protocols.

  • Test different antigen retrieval methods: heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0), as well as enzymatic retrieval with proteinase K.

  • Optimize retrieval duration and temperature based on tissue type and fixation conditions.

• Antibody selection and dilution:

  • Choose antibodies specifically validated for IHC applications .

  • Test a range of antibody dilutions (typically starting with 1:50-1:200) to determine optimal signal-to-noise ratio.

  • Consider using both N-terminal and C-terminal antibodies in parallel sections to confirm specificity .

• Blocking and incubation conditions:

  • Use appropriate blocking solutions containing serum matched to the secondary antibody host species.

  • Optimize primary antibody incubation time and temperature (4°C overnight often yields better results than shorter incubations at room temperature).

  • Include 0.1-0.3% Triton X-100 in buffers if improved penetration is needed.

• Detection system selection:

  • For low abundance proteins like APC10, amplification systems such as tyramide signal amplification may improve detection sensitivity.

  • Choose detection systems compatible with your microscopy setup (chromogenic vs. fluorescent).

• Counter-staining and mounting:

  • Select counterstains that don't interfere with APC10 detection (e.g., hematoxylin for chromogenic detection).

  • Use appropriate antifade mounting media for fluorescent detection to prevent photobleaching.

• Validation of staining patterns:

  • Compare staining patterns with published localization data (primarily cytoplasmic/apical distribution for APC10) .

  • Confirm specificity through appropriate controls as discussed in question 3.2.

These optimization steps will help achieve consistent and specific APC10 detection in tissue samples.

What are the recommended approaches for studying APC10's role in cell cycle regulation?

To effectively study APC10's role in cell cycle regulation, researchers can employ several methodological approaches using APC10 antibodies:

• Cell synchronization experiments:

  • Synchronize cells at different cell cycle phases (G1/S using double thymidine block, M phase using nocodazole).

  • Analyze APC10 expression, localization, and interaction partners across these phases using western blotting, immunofluorescence, and co-immunoprecipitation with APC10 antibodies.

• APC/C substrate degradation assays:

  • Monitor the levels of known APC/C substrates (e.g., securin, cyclin B) in relation to APC10 manipulation (knockdown/overexpression).

  • Use APC10 antibodies to confirm knockdown efficiency or overexpression levels.

• Ubiquitination assays:

  • Perform in vitro or in vivo ubiquitination assays to assess how APC10 manipulation affects substrate ubiquitination.

  • Studies have shown that Apc10 is required for the ubiquitination of Cdc13 (a cyclin), and this can be demonstrated using proteasome mutant backgrounds to accumulate ubiquitinated intermediates .

• Live-cell imaging:

  • Combine APC10 immunostaining with live-cell imaging to track cell cycle progression.

  • Correlate APC10 localization or activity with specific cell cycle events.

• Interaction mapping:

  • Use co-immunoprecipitation with APC10 antibodies followed by mass spectrometry to identify novel APC10 interaction partners during different cell cycle phases.

  • Investigate APC10's physical interaction with other APC/C subunits, such as the demonstrated interaction with Nuc2 .

• Functional rescue experiments:

  • In APC10-depleted cells, reintroduce wild-type or mutant APC10 constructs and analyze their ability to restore normal cell cycle progression.

  • Use APC10 antibodies to confirm expression levels of the rescue constructs.

These approaches, often used in combination, can provide comprehensive insights into APC10's functions in cell cycle regulation and substrate recognition within the APC/C complex.

How are APC10 antibodies being used to study the dual role of APC10 in cell division and inflammation?

Recent research has uncovered a fascinating connection between APC10's canonical role in cell cycle regulation and a novel function in inflammatory responses . APC10 antibodies are instrumental in studying this dual functionality:

• Cell cycle-dependent regulation of inflammasome activation:

  • Immunofluorescence studies using APC10 antibodies have revealed that during interphase, APC10 interacts with NLRP3 to promote inflammasome activation, whereas during mitosis, this interaction is disrupted .

  • Co-immunoprecipitation experiments with APC10 antibodies help determine the cell cycle-dependent changes in APC10-NLRP3 interactions.

• Spatial and temporal regulation:

  • Super-resolution microscopy combined with APC10 immunostaining helps visualize the dynamic localization of APC10 relative to inflammasome components throughout the cell cycle.

  • Time-lapse imaging with fluorescently labeled antibody fragments can track real-time changes in these interactions.

• Mechanistic investigations:

  • APC10 antibodies used in ChIP-seq (Chromatin Immunoprecipitation Sequencing) experiments may reveal whether APC10 influences the expression of inflammasome components.

  • Proximity-dependent biotinylation (BioID) combined with APC10 antibody validation helps identify additional proteins that mediate the APC10-inflammasome connection.

• Pathological relevance:

  • Immunohistochemistry with APC10 antibodies in tissues from inflammatory disease models helps assess whether dysregulation of this dual role contributes to pathological inflammation.

  • Flow cytometry with APC10 and inflammasome component antibodies allows correlation between cell cycle phase and inflammatory activation at the single-cell level.

This emerging research area highlights how APC10 serves as a critical link between cell proliferation and innate immune responses, with important implications for understanding inflammatory disorders .

What experimental considerations are important when using APC10 antibodies for protein degradation pathway studies?

APC10's role in the APC/C E3 ubiquitin ligase complex makes it a valuable target for studying protein degradation pathways. When using APC10 antibodies in these studies, consider:

• Proteasome inhibition strategies:

  • Combine APC10 antibody detection with proteasome inhibitors (MG132, bortezomib) to accumulate ubiquitinated substrates.

  • Studies using proteasome mutant backgrounds have demonstrated that Apc10 is required for the ubiquitination of substrates like Cdc13 .

• Ubiquitination detection methods:

  • Use APC10 antibodies for immunoprecipitation followed by ubiquitin antibody detection to identify ubiquitinated APC10 substrates.

  • Consider using tandem ubiquitin-binding entities (TUBEs) to enrich ubiquitinated proteins before APC10 antibody analysis.

• Substrate recognition analysis:

  • APC10 plays a critical role in substrate recognition within the APC/C complex .

  • Use site-directed mutagenesis of APC10 combined with antibody detection to identify regions crucial for substrate binding.

• APC/C complex integrity assessment:

  • Co-immunoprecipitation with APC10 antibodies can help determine whether experimental manipulations affect APC/C complex assembly.

  • Gradient centrifugation followed by western blotting with APC10 antibodies can reveal changes in complex formation.

• Cell-free degradation assays:

  • In vitro reconstitution of the ubiquitination machinery using purified components and verification with APC10 antibodies.

  • Cell-free extracts from synchronized cells can be used to study cell cycle-dependent degradation, with APC10 antibodies confirming the presence of active APC/C.

• Comparative analysis across model systems:

  • APC10 antibodies that cross-react with multiple species (human, mouse, rat) allow for evolutionary conservation studies of degradation mechanisms .

These considerations help researchers design rigorous experiments to elucidate the mechanisms of APC10-mediated protein degradation and its regulation in various biological contexts.

What emerging techniques incorporate APC10 antibodies for studying protein dynamics in live cells?

• Antibody-based biosensors:

  • Fluorescently labeled antibody fragments (Fabs) or single-chain variable fragments (scFvs) derived from APC10 antibodies can be introduced into live cells to track APC10 dynamics.

  • FRET-based biosensors incorporating APC10 antibody-derived recognition domains can report on conformational changes or protein-protein interactions.

• Nanobody technology:

  • Camelid-derived single-domain antibodies (nanobodies) against APC10 can be expressed intracellularly as fusion proteins with fluorescent tags, allowing real-time visualization with minimal disruption to protein function.

  • These can be combined with optogenetic tools to manipulate APC10 function with spatiotemporal precision.

• Split-antibody complementation systems:

  • Engineer complementary fragments of fluorescent proteins fused to APC10 antibody-derived binding domains that reconstitute fluorescence only when APC10 interacts with binding partners.

• Advanced microscopy techniques:

  • Single-molecule tracking using quantum dot-conjugated APC10 antibody fragments to follow individual APC10 molecules within living cells.

  • Lattice light-sheet microscopy combined with APC10 antibody probes for high-speed, low-phototoxicity 3D imaging of APC10 dynamics during cell division.

• Correlative light and electron microscopy (CLEM):

  • Use fluorescently labeled APC10 antibodies for live-cell imaging followed by electron microscopy of the same cells to correlate functional dynamics with ultrastructural context.

• Protein-retention expansion microscopy (ProExM):

  • Apply APC10 antibodies in physically expanded cell samples to achieve super-resolution imaging on conventional microscopes.

These emerging techniques promise to provide unprecedented insights into APC10's dynamic functions in cell cycle regulation and inflammatory processes, bridging the gap between fixed-cell analyses and live-cell protein dynamics.

How can researchers validate novel APC10 antibodies for specific research applications?

Validating novel APC10 antibodies requires a systematic approach to ensure specificity, sensitivity, and reproducibility:

• Comprehensive characterization table:

• Application-specific validation:

• For western blotting: Determine optimal sample preparation conditions (lysis buffers, reducing vs. non-reducing conditions), working dilutions, and exposure times.

• For immunofluorescence: Optimize fixation methods (paraformaldehyde, methanol, or glutaraldehyde), permeabilization conditions, and antibody concentrations.

• For immunoprecipitation: Test different binding conditions, wash stringency, and elution methods.

• For ELISA: Establish standard curves, determine detection limits, and assess cross-reactivity with related proteins.

• Physiological validation:

• Test antibody performance across different cell cycle phases when APC10 activity naturally varies.

• Examine detection in different cell types with varying APC10 expression levels.

• Verify expected subcellular localization patterns against published data (typically cytoplasmic/apical distribution) .

• Reproducibility assessment:

• Evaluate lot-to-lot consistency if producing custom antibodies.

• Test performance across different sample types (cell lines, primary cells, tissue sections).

• Document detailed protocols to ensure reproducibility across laboratory members and collaborative studies.

This systematic validation approach ensures that novel APC10 antibodies will generate reliable and interpretable data for the intended research applications.

What are the key considerations for selecting the most appropriate APC10 antibody for specific research questions?

Selecting the optimal APC10 antibody requires careful consideration of several factors to ensure experimental success:

• Match antibody characteristics to your research application: Different applications (western blotting, immunofluorescence, immunoprecipitation) may require antibodies with distinct properties. Review validation data for your specific application .

• Consider epitope location: N-terminal versus C-terminal antibodies may yield different results, especially when studying potential truncated variants or when protein interactions might mask certain epitopes .

• Evaluate species reactivity: Ensure the antibody recognizes APC10 in your model organism. Many antibodies show cross-reactivity with human, mouse, and rat APC10, but verify this for your specific research needs .

• Assess clonality requirements: Polyclonal antibodies often provide higher sensitivity but potentially lower specificity compared to monoclonals. For critical quantitative applications, monoclonal antibodies may offer better reproducibility .

• Be aware of known cross-reactivity: Several APC10 antibodies recognize a consistent 150 kDa protein that is unlikely to be APC10. Understanding such limitations helps interpret experimental results accurately .

• Consider functional studies: For research exploring APC10's role in specific pathways (cell cycle regulation, inflammasome activation), select antibodies validated in similar functional contexts .

• Verify compatibility with experimental conditions: Some antibodies perform differently under various fixation, lysis, or buffer conditions. Match these parameters to your experimental design.