APC13 Antibody

What is the function of ANAPC13 protein in cell cycle regulation?

ANAPC13 functions as a component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated E3 ubiquitin ligase that controls progression through mitosis and the G1 phase of the cell cycle. Specifically, APC13 promotes the stable association of APC3/Cdc27 and APC6/Cdc16 with the APC/C complex . The APC/C complex mediates ubiquitination and subsequent degradation of target proteins, primarily through the formation of 'Lys-11'-linked polyubiquitin chains and, to a lesser extent, 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains . Notably, the APC/C complex catalyzes the assembly of branched 'Lys-11'-/'Lys-48'-linked ubiquitin chains on target proteins, marking them for degradation and thereby regulating cell cycle progression .

What are the recommended applications for APC13 antibody?

APC13 antibody has been validated for multiple experimental applications with specific concentration recommendations:

The antibody's species reactivity includes human, mouse, and rat samples . For optimal results, researchers should titrate the antibody carefully for each specific application and experimental system, as different cellular contexts may require adjusted concentrations.

How should APC13 antibody be stored to maintain its effectiveness?

Proper storage of APC13 antibody is crucial for maintaining its reactivity and specificity. The antibody can be stored at 4°C for up to three months for ongoing experiments . For long-term storage, maintain the antibody at -20°C, where it remains stable for up to one year . To preserve antibody function, avoid repeated freeze-thaw cycles as these can lead to protein denaturation and decreased antibody efficacy. It's recommended to aliquot the antibody upon receipt to minimize the number of freeze-thaw cycles. Additionally, antibodies should not be exposed to prolonged high temperatures as this can accelerate degradation and reduce binding capacity .

What controls should be included when using APC13 antibody for Western blot experiments?

When designing Western blot experiments with APC13 antibody, include the following controls to ensure reliable and interpretable results:

• Positive Control: Cell lysates known to express APC13/ANAPC13 (such as HeLa or HEK293 cells) to confirm antibody functionality.

• Negative Control: Either:

  • Lysates from cells where APC13 has been knocked down via RNAi

  • Tissue lysates that do not express APC13

  • Secondary antibody-only control to identify non-specific binding

• Loading Control: Include detection of a housekeeping protein (β-actin, GAPDH) to normalize protein loading.

• Molecular Weight Marker: Use to confirm the detection of APC13 at its calculated molecular weight of 8.5 kDa .

• Peptide Competition Assay: Pre-incubation of the antibody with the immunizing peptide should abolish specific signal, validating antibody specificity.

This comprehensive approach mirrors validation methods used for other APC-related antibodies and helps distinguish true signal from potential cross-reactivity, which has been documented as a concern with some antibodies in this family .

How can I optimize immunofluorescence protocols using APC13 antibody?

Optimizing immunofluorescence with APC13 antibody requires attention to several key parameters:

• Initial Concentration: Begin with the recommended 20 μg/mL concentration, but be prepared to titrate to determine optimal signal-to-noise ratio .

• Fixation Method:

  • For nuclear proteins like APC13, 4% paraformaldehyde (10-15 minutes) typically preserves structure while maintaining antibody epitope accessibility.

  • Compare with methanol fixation (-20°C for 10 minutes) which may enhance nuclear protein detection.

• Permeabilization: Use 0.1-0.5% Triton X-100 in PBS for 5-10 minutes to ensure antibody access to nuclear targets.

• Blocking: Incubate with 5% normal serum (matching secondary antibody host) with 0.1% Triton X-100 for 30-60 minutes.

• Primary Antibody Incubation: Test both room temperature (1-2 hours) and 4°C overnight incubations to determine optimal binding conditions.

• Signal Amplification: For weak signals, consider tyramide signal amplification or use of high-sensitivity detection systems.

• Validation: Include a negative control using RNAi knockdown of APC13 to confirm specificity of observed staining patterns .

Remember that subcellular localization is primarily nuclear for APC13, which should guide your expected staining pattern interpretation.

Why might I detect bands other than the expected 8.5 kDa APC13 protein in Western blot?

The detection of unexpected bands when using APC13 antibody warrants careful analysis, as similar issues have been documented with other APC antibodies . Potential causes include:

• Post-translational Modifications: Phosphorylation, ubiquitination, or other modifications can alter protein migration, resulting in shifted bands.

• Cross-Reactivity: The antibody may recognize epitopes present in other proteins. This is particularly relevant for polyclonal antibodies like the APC13 antibody, which contain multiple antibody clones recognizing different epitopes .

• Protein Degradation: Incomplete protease inhibition during sample preparation may lead to degradation products appearing as lower molecular weight bands.

• Protein Complexes: Incompletely denatured protein complexes may appear as higher molecular weight bands.

• Non-specific Binding: Secondary antibody binding to endogenous immunoglobulins or Fc receptors.

To address these issues:

• Use fresh samples with complete protease inhibitor cocktails

• Optimize sample denaturation conditions

• Perform peptide competition assays to identify specific binding

• Consider using knockout/knockdown controls to validate band specificity

• Compare results with alternative antibodies targeting different epitopes of APC13

Research has shown that some antibodies targeting APC family proteins consistently detect non-specific proteins (e.g., a 150 kDa protein detected by multiple APC antibodies) , emphasizing the importance of thorough validation.

How can I quantitatively assess APC13 levels in different cell cycle phases?

To quantitatively measure APC13 levels across different cell cycle phases, combine the following approaches:

• Cell Synchronization and Western Blot Analysis:

  • Synchronize cells using standard methods (double thymidine block, nocodazole arrest, etc.)

  • Collect samples at defined time points as cells progress through the cycle

  • Perform Western blot with APC13 antibody (1 μg/mL)

  • Quantify band intensities using image analysis software

  • Normalize to loading controls

  • Correlate with cell cycle markers (Cyclin B1, phospho-histone H3, etc.)

• Flow Cytometry Approach:

  • Fix cells with 70% ethanol (drop-wise while vortexing)

  • Permeabilize with 0.25% Triton X-100

  • Incubate with APC13 antibody followed by fluorescent secondary antibody

  • Counterstain with propidium iodide for DNA content

  • Analyze correlation between APC13 signal intensity and cell cycle phase

• Immunofluorescence Microscopy with Cell Cycle Markers:

  • Co-stain cells with APC13 antibody (20 μg/mL) and cell cycle phase markers

  • Capture images using consistent exposure settings

  • Measure nuclear fluorescence intensity with image analysis software

  • Categorize cells by cycle phase based on marker expression

  • Compare APC13 intensities across phases

This multi-technique approach provides complementary quantitative data on APC13 dynamics throughout the cell cycle, offering insights into its regulation and function.

How can I investigate the role of APC13 in the assembly and activity of the APC/C complex?

Investigating APC13's role in APC/C complex assembly and activity requires multiple complementary approaches:

• Co-immunoprecipitation Studies:

  • Use APC13 antibody for immunoprecipitation from cell lysates

  • Analyze precipitated proteins by Western blot for APC/C components (APC3/Cdc27, APC6/Cdc16)

  • Compare complex composition between wild-type and APC13-depleted cells

  • Assess how complex formation changes during cell cycle progression

• Proximity Ligation Assay (PLA):

  • Perform PLA using APC13 antibody and antibodies against other APC/C components

  • Quantify interaction signals in different cell cycle phases

  • Compare interaction patterns in normal vs. stressed conditions

• In Vitro Ubiquitination Assays:

  • Purify APC/C from cells with normal or depleted APC13 levels

  • Measure ubiquitination activity toward known substrates (securin, cyclin B)

  • Analyze ubiquitin chain types formed (Lys-11, Lys-48, branched chains)

  • Test if recombinant APC13 can restore activity to depleted complexes

• Cross-linking Mass Spectrometry:

  • Cross-link purified APC/C complexes

  • Digest and analyze by mass spectrometry

  • Map APC13 interaction surfaces with APC3/Cdc27 and APC6/Cdc16

  • Create structural models of APC13's position within the complex

This comprehensive approach will provide mechanistic insights into how APC13 promotes the stable association of key components within the APC/C complex and influences its ubiquitination activity.

What are the considerations for using APC13 antibody in studying patient-derived cancer samples?

When applying APC13 antibody to cancer research using patient-derived samples, several important considerations must be addressed:

• Sample Preparation Optimization:

  • Fresh frozen vs. FFPE tissue samples may require different antibody concentrations

  • Antigen retrieval methods should be carefully optimized for FFPE samples

  • Patient-derived xenografts may require species-specific secondary antibodies to avoid cross-reactivity

• Validation in Relevant Cancer Models:

  • Before analyzing patient samples, validate antibody performance in cell lines representing the cancer type

  • Confirm specificity using genetic approaches (CRISPR/RNAi) in these models

  • Establish interpretation guidelines based on known APC/C dysregulation patterns

• Correlation with Clinical Parameters:

  • Design studies to correlate APC13 expression/localization with:

    • Tumor grade and stage

    • Proliferation markers (Ki-67, mitotic index)

    • Patient outcomes and treatment response

  • Use multivariate analysis to distinguish APC13-specific effects

• Technical Considerations:

  • Include multiple normal tissue controls from the same organ

  • Implement tissue microarrays for standardized high-throughput analysis

  • Consider multiplex immunofluorescence to analyze APC13 in relation to other APC/C components or cell cycle markers

• Potential Confounding Factors:

  • APC13 antibody might cross-react with other proteins, as observed with other APC antibodies

  • Cancer-specific post-translational modifications might affect epitope recognition

  • Heterogeneity within tumors may require analysis of multiple regions

This approach ensures that findings related to APC13 in cancer samples are robust, reproducible, and clinically relevant, potentially revealing new insights into cell cycle dysregulation in cancer.

How can I develop assays to screen for compounds that modulate APC13 interaction with the APC/C complex?

Developing high-throughput assays to identify compounds affecting APC13-APC/C interactions requires sophisticated methodological approaches:

• FRET-based Interaction Assays:

  • Generate fusion proteins: APC13-CFP and APC3/Cdc27-YFP or APC6/Cdc16-YFP

  • Express in appropriate cell lines or in vitro translation systems

  • Measure FRET signal as readout of protein proximity

  • Screen compounds for ability to disrupt or enhance FRET signal

  • Validate hits using orthogonal methods

• AlphaScreen/AlphaLISA Technology:

  • Couple purified APC13 and APC3/Cdc27 to donor and acceptor beads

  • Measure luminescence signal indicating protein interaction

  • Test compound libraries in 384/1536-well format

  • Identify compounds that alter signal intensity

  • Determine dose-response relationships

• Split Luciferase Complementation:

  • Create APC13-NLuc and APC6-CLuc fusion constructs

  • Express in cells and measure reconstituted luciferase activity

  • Screen for compounds that modulate luminescence

  • Establish cell-based validation assays for promising hits

• Surface Plasmon Resonance (SPR):

  • Immobilize purified APC13 on sensor chip

  • Measure binding kinetics with APC3/Cdc27 and APC6/Cdc16

  • Test compounds for ability to alter binding parameters

  • Determine binding constants and competition mechanisms

• Cellular Phenotype Readouts:

  • Design reporter cell lines where APC/C activity drives fluorescent protein expression

  • Use high-content imaging to measure effects on cell cycle progression

  • Correlate with biochemical assays of APC13-APC/C interaction

These methodologies provide complementary approaches to identify compounds that could serve as chemical probes for studying APC13 function or as starting points for therapeutic development targeting the APC/C complex in diseases like cancer.

How can new antibody technologies improve APC13 detection and functional studies?

Emerging antibody technologies offer significant advantages for APC13 research compared to traditional polyclonal antibodies:

• Single-Domain Antibodies (Nanobodies):

  • Smaller size (~15 kDa vs ~150 kDa for conventional antibodies) allows better penetration of dense structures

  • Can access cryptic epitopes that might be inaccessible to larger antibodies

  • Potential for intracellular expression as "intrabodies" to track APC13 in living cells

  • Development through microfluidics-enabled approaches for faster discovery

• Site-Specific Conjugation Technologies:

  • Precise addition of fluorophores or enzymes at defined positions

  • Maintains antibody orientation for optimal epitope binding

  • Reduces batch-to-batch variability compared to random chemical conjugation

  • Enables quantitative super-resolution microscopy of APC13 within the APC/C complex

• Recombinant Antibody Fragments:

  • Fab, scFv, or Fab2 formats with improved tissue penetration

  • Genetically encoded for consistent performance across experiments

  • Potential for rational engineering to improve affinity and specificity

  • Compatible with phage display for epitope-specific selection

• Multiplexed Detection Systems:

  • DNA-barcoded antibodies for simultaneous detection of multiple APC/C components

  • Mass cytometry (CyTOF) using metal-tagged antibodies for high-parameter analysis

  • Proximity extension assays for ultrasensitive detection of low-abundance modifications

• Validation Technologies:

  • CRISPR knockout validation to definitively confirm specificity

  • Automated microfluidic platforms for systematic antibody characterization

  • Standardized validation protocols across different applications

These technologies address the limitations of traditional antibodies and enable more precise, quantitative, and mechanistic studies of APC13's role in cell cycle regulation and cancer biology.

What are the considerations for studying post-translational modifications of APC13 using specific antibodies?

Studying post-translational modifications (PTMs) of APC13 presents unique challenges that require specialized approaches:

• Identification of Relevant Modifications:

  • Perform mass spectrometry analysis to identify PTMs on APC13

  • Focus on modifications that change during cell cycle or in response to cellular stress

  • Prioritize sites conserved across species for functional significance

  • Generate modification site maps in relation to APC13's functional domains

• Development of Modification-Specific Antibodies:

  • Design peptide antigens containing the specific modification

  • Implement strict validation protocols including:

    • Peptide competition with modified vs. unmodified peptides

    • Testing on samples with enzymes that remove the specific modification

    • Validation in cells with mutation of the modified residue

• Quantitative Analysis Methods:

  • Develop ELISA or AlphaLISA assays for specific modifications

  • Establish Western blot protocols with modification-specific antibodies

  • Implement multiplexed assays to simultaneously measure multiple PTMs

  • Create standardized curves using recombinant proteins with defined modifications

• Functional Analysis Approaches:

  • Correlate modification levels with APC/C activity using in vitro ubiquitination assays

  • Investigate cell cycle-dependent changes in modifications

  • Study how modifications affect APC13's interaction with APC3/Cdc27 and APC6/Cdc16

  • Generate modification-mimicking or modification-blocking mutations for in vivo studies

• Technical Considerations and Challenges:

  • Low abundance of APC13 may require enrichment strategies before analysis

  • Modification site accessibility might be limited within the APC/C complex

  • Multiple modifications might occur simultaneously with combinatorial effects

  • Antibody cross-reactivity with similar modification sites on other proteins

This systematic approach enables researchers to understand how PTMs regulate APC13 function, potentially revealing new mechanisms of APC/C regulation and identifying novel intervention points for therapeutic development.

What quality control measures should researchers implement when using APC13 antibodies in long-term projects?

Implementing rigorous quality control for APC13 antibodies in longitudinal studies is essential for generating reliable and reproducible data:

• Antibody Validation Documentation:

  • Maintain detailed records of initial validation experiments

  • Document lot-to-lot testing results and comparisons

  • Create laboratory-specific validation protocols based on experimental applications

  • Establish minimum performance criteria for each application

• Regular Performance Assessment:

  • Test each new antibody lot against a reference standard

  • Maintain frozen aliquots of positive control samples for consistency

  • Implement quantitative metrics for signal-to-noise ratio evaluation

  • Compare antibody performance across different detection systems

• Storage and Handling Protocols:

  • Establish strict temperature monitoring for antibody storage

  • Create working aliquots to minimize freeze-thaw cycles

  • Document antibody age and use history

  • Maintain standardized dilution and preparation methods

• Controls for Experimental Variation:

  • Include consistent positive and negative controls in each experiment

  • Consider using recombinant APC13 as a standard reference

  • Implement spike-in controls for quantitative applications

  • Use multiple antibodies targeting different epitopes when possible

• Addressing Issues with Cross-Reactivity:

  • Regular revalidation using knockdown/knockout approaches

  • Peptide competition assays to confirm specificity

  • Continuous monitoring of literature for reports of cross-reactivity

  • Development of orthogonal detection methods

These systematic quality control measures ensure consistent antibody performance throughout long-term projects and facilitate reliable comparison of data collected over extended time periods, addressing known concerns about antibody specificity in the APC family .

How might APC13 antibodies contribute to understanding cell cycle dysregulation in cancer and other diseases?

APC13 antibodies serve as critical tools for elucidating the role of the APC/C complex in disease states:

• Diagnostic and Prognostic Applications:

  • Development of immunohistochemistry protocols for patient stratification

  • Correlation of APC13 levels or localization with disease progression

  • Integration with other cell cycle markers for comprehensive profiling

  • Potential identification of APC13 alterations as biomarkers

• Mechanistic Studies in Cancer Models:

  • Investigation of APC/C complex integrity in various cancer types

  • Analysis of APC13's role in maintaining genomic stability

  • Evaluation of how oncogenic signaling affects APC13 function

  • Examination of APC13's contribution to chemotherapy response

• Therapeutic Target Validation:

  • Use of APC13 antibodies to identify protein-protein interaction sites

  • Development of proximity-based assays to screen for disruptors or enhancers

  • Monitoring of APC/C activity in response to cell cycle-targeting drugs

  • Assessment of APC13 as a potential vulnerability in cancer cells

• System-level Analysis of Cell Cycle Regulation:

  • Integration of APC13 data with other APC/C components

  • Mapping of dynamic changes in complex composition during disease progression

  • Correlation with ubiquitination patterns of downstream targets

  • Computational modeling of APC/C dysregulation in disease states

• Non-canonical Functions Exploration:

  • Investigation of potential roles beyond canonical cell cycle regulation

  • Analysis in non-proliferating specialized cells

  • Examination of tissue-specific functions and interactions

  • Exploration of roles in other cellular processes like DNA damage response