ANAPC1 Antibody

What is ANAPC1 and what cellular functions does it serve?

ANAPC1, also known as mitotic checkpoint regulator (MCPR), is a highly conserved component of the anaphase promoting complex/cyclosome (APC/C). This cell cycle-regulated E3 ubiquitin ligase controls progression through mitosis and the G1 phase of the cell cycle . The APC/C complex, which includes ANAPC1, is responsible for degrading anaphase inhibitors, mitotic cyclins, and spindle-associated proteins to ensure proper sequential progression of mitotic events . Individual APC/C components, including ANAPC1, are expressed at relatively consistent levels across most tissues and cell lines, suggesting they primarily function as part of this complex rather than independently .

What are the common applications for ANAPC1 antibodies in experimental research?

ANAPC1 antibodies have been validated for multiple experimental applications, with different antibodies showing varying effectiveness across techniques. The most common applications include:

When designing experiments, researchers should select antibodies specifically validated for their intended application to ensure optimal results .

What considerations are important when selecting between polyclonal and monoclonal ANAPC1 antibodies?

Most commercially available ANAPC1 antibodies are polyclonal, including those referenced in the search results. Polyclonal antibodies recognize multiple epitopes on the ANAPC1 protein, providing advantages for certain applications:

• Detection sensitivity is typically higher as multiple epitopes are recognized

• More robust against minor protein conformational changes

• Often more effective for applications like immunoprecipitation

The polyclonal antibodies in the search results were developed using different methodological approaches:

• Some were raised against fusion proteins (BS-4090R, 21748-1-AP)

• Others target specific phosphorylation sites (pSer377)

• Some target specific amino acid sequences (AA 373-382)

When selecting an ANAPC1 antibody, researchers should evaluate whether their experimental question requires detection of specific post-translational modifications, particular protein domains, or total ANAPC1 protein levels .

What positive controls are recommended for validating ANAPC1 antibodies?

Based on validation data in the search results, the following samples serve as effective positive controls for ANAPC1 antibody testing:

Including appropriate positive controls is essential for confirming antibody specificity and optimizing experimental conditions .

How does ANAPC1 expression correlate with cancer progression, and what implications does this have for research applications?

Recent comprehensive analysis of 2,031 samples revealed significant upregulation of ANAPC1 mRNA in lung squamous cell carcinoma (LUSC) tissues (SMD = 1.97, 95% CI [1.26–2.67]) . This finding was further confirmed at the protein level through immunohistochemical analysis . The clinical significance of this overexpression is substantial:

Functionally, ANAPC1 knockdown inhibits cell proliferation, while overexpression reduces immune cell infiltration and immunotherapy effectiveness . These findings suggest ANAPC1 antibodies are valuable tools for cancer research, particularly in:

• Prognostic biomarker development

• Therapeutic target validation

• Patient stratification studies

• Mechanistic investigations of cell cycle dysregulation in cancer

What methodological considerations are critical when using phospho-specific ANAPC1 antibodies?

Phosphorylation may play a crucial regulatory role in ANAPC1 function. When using phospho-specific antibodies such as those targeting pSer377 , researchers should consider:

• Sample preparation protocols:

  • Rapid sample collection and processing is essential

  • Include phosphatase inhibitors in all buffers

  • Avoid freeze-thaw cycles that may alter phosphorylation states

• Validation requirements:

  • Confirm specificity using phosphatase-treated negative controls

  • Verify phospho-specificity with competing phospho-peptides

  • Test cross-reactivity with non-phosphorylated ANAPC1 protein

• Experimental design:

  • Consider cell cycle synchronization to capture dynamic phosphorylation events

  • Evaluate phosphorylation under different cellular stresses or treatments

  • Correlate phosphorylation with functional outcomes

• Storage considerations:

  • Aliquot antibodies to avoid freeze-thaw cycles

  • Store at -20°C as recommended for phospho-specific antibodies

  • Consider adding carriers like BSA for dilute solutions

What strategies can improve detection of high molecular weight ANAPC1 protein in Western blot applications?

ANAPC1 is a large protein with a calculated molecular weight of 217 kDa (1944 amino acids), though it typically appears at 200-210 kDa on Western blots . This size creates technical challenges requiring specific optimization:

• Sample preparation:

  • Use strong lysis buffers (RIPA or stronger) with protease inhibitors

  • Extend denaturation time (10+ minutes at 95°C)

  • Sonicate samples to shear DNA and improve protein extraction

• Gel electrophoresis:

  • Use low percentage acrylamide gels (6-8%) or gradient gels

  • Run at lower voltage for longer time to improve separation

  • Include high molecular weight markers

• Transfer optimization:

  • Extend transfer time (overnight at low voltage is often optimal)

  • Reduce methanol concentration in transfer buffer

  • Consider semi-dry transfer systems designed for large proteins

• Detection protocols:

  • Use recommended dilution range (1:1000-1:4000)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Use high-sensitivity detection systems

• Troubleshooting approach:

  • Begin with validated positive controls (HeLa cells, HEK-293 cells)

  • Compare results across different antibody concentrations

  • Consider blocking with both milk and BSA to determine optimal conditions

How can researchers optimize immunohistochemistry protocols for ANAPC1 detection in different tissue types?

Optimizing IHC protocols for ANAPC1 detection requires attention to several technical aspects:

• Antigen retrieval methods:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative approach: citrate buffer pH 6.0

  • Extended retrieval times (20-30 minutes) may improve detection of nuclear proteins

• Antibody dilution optimization:

  • Begin with manufacturer recommended range (1:50-1:500)

  • Perform dilution series to identify optimal signal-to-noise ratio

  • Consider tissue-specific optimization (different optimal dilutions for different tissues)

• Signal amplification considerations:

  • Standard avidin-biotin complex methods work well for most applications

  • Tyramide signal amplification may improve detection of low abundance targets

  • Polymer detection systems can reduce background in challenging tissues

• Tissue-specific considerations:

  • Successfully tested tissues include: mouse testis, human cervical cancer, human stomach

  • Different fixation protocols may require adjusted antigen retrieval methods

  • Consider double staining with cell-type markers to confirm cellular localization

• Control selection:

  • Positive control: mouse testis tissue shows reliable ANAPC1 expression

  • Negative control: omit primary antibody but perform all other steps

  • Competing peptide control: pre-incubate antibody with immunizing peptide

How can researchers effectively study the role of ANAPC1 in APC/C complex formation and cell cycle regulation?

Understanding ANAPC1's role in the APC/C complex requires sophisticated experimental approaches:

• Co-immunoprecipitation studies:

  • Use ANAPC1 antibodies to pull down the intact complex

  • Western blot for other APC/C components to confirm association

  • Compare complex composition across cell cycle phases

• Proximity-based interaction assays:

  • Proximity ligation assay (PLA) to visualize interactions in situ

  • FRET/BRET approaches for real-time interaction dynamics

  • BioID or APEX2 proximity labeling to identify novel interactions

• Functional assays:

  • Substrate ubiquitination assays with reconstituted complexes

  • Cell cycle progression analysis after ANAPC1 depletion/mutation

  • Live cell imaging with fluorescently tagged ANAPC1 and substrates

• Structural approaches:

  • Cryo-EM analysis of complete APC/C complex

  • Domain mapping through deletion/mutation analysis

  • Cross-linking mass spectrometry to identify interaction interfaces

• Cell cycle synchronization methods:

  • Double thymidine block for G1/S boundary

  • Nocodazole treatment for M phase

  • Analyze ANAPC1 expression, localization and modification across phases

The highly conserved nature of ANAPC1 across species suggests evolutionary importance in APC/C function , making comparative studies across model organisms potentially informative.

What approaches can researchers use to investigate interactions between ANAPC1 and microRNAs in disease contexts?

Recent research has identified interactions between ANAPC1 and microRNAs in disease contexts . To investigate these interactions, researchers can employ several methodological approaches:

• Expression correlation analysis:

  • Quantify ANAPC1 and candidate miRNAs across patient samples

  • Perform correlation analysis to identify significant associations

  • Stratify by disease subtype, stage, or outcome

• Direct binding assessment:

  • Luciferase reporter assays with ANAPC1 3'UTR constructs

  • miRNA mimic/inhibitor transfection followed by ANAPC1 quantification

  • RNA immunoprecipitation to capture ANAPC1 mRNA-miRNA complexes

• Functional validation:

  • CRISPR-mediated mutation of miRNA binding sites in ANAPC1

  • Rescue experiments with miRNA-resistant ANAPC1 constructs

  • Phenotypic assays following miRNA or ANAPC1 modulation

• Clinical relevance assessment:

  • Patient sample analysis stratified by ANAPC1/miRNA expression patterns

  • Survival analysis based on ANAPC1/miRNA signatures

  • Development of multivariate prediction models incorporating both factors

• Therapeutic targeting strategies:

  • miRNA delivery approaches to modulate ANAPC1 expression

  • Small molecule screens to identify compounds disrupting miRNA-ANAPC1 interactions

  • Combination approaches targeting both ANAPC1 and associated miRNAs

How can researchers optimize detection of ANAPC1 in rapid antibody discovery platforms using microfluidics?

Emerging microfluidics-enabled antibody discovery platforms can be adapted for ANAPC1 antibody development or screening:

• Single-cell compartmentalization strategy:

  • Encapsulate antibody-secreting cells in hydrogel beads

  • Maintain the crucial genotype-phenotype link between cell and secreted antibody

  • Process millions of cells per hour using droplet microfluidics

• Detection optimization:

  • Label recombinant ANAPC1 protein with fluorescent markers

  • Implement multi-parameter detection to assess binding specificity

  • Use FACS for high-throughput screening of binding events

• Validation approach:

  • Secondary screening for cross-reactivity with related proteins

  • Epitope binning to identify antibodies targeting different regions

  • Functional assays to identify antibodies that modulate ANAPC1 activity

• Technical considerations:

  • Optimize secretion time (1-2 hours recommended based on similar approaches)

  • Balance sensitivity and throughput in platform design

  • Include controls for non-specific binding to hydrogel matrix

• Application-specific optimization:

  • For phospho-specific antibodies, use phosphorylated peptides as targets

  • For domain-specific antibodies, use truncated protein constructs

  • For neutralizing antibodies, implement functional screening component

This microfluidics approach can dramatically accelerate ANAPC1 antibody development, reducing traditional timeframes from months to weeks .

What methodological approaches can resolve contradictory experimental results when studying ANAPC1 in different cancer contexts?

When faced with contradictory results regarding ANAPC1's role in different cancer contexts, researchers should consider:

• Systematic validation approach:

  • Use multiple antibodies targeting different ANAPC1 epitopes

  • Employ both RNA and protein detection methods

  • Include appropriate positive and negative controls

• Technical variation analysis:

  • Standardize sample preparation, fixation, and processing

  • Compare antibody performance across different batches

  • Implement quantitative analysis methods with normalization

• Biological context evaluation:

  • Assess cell type-specific ANAPC1 functions

  • Consider tumor heterogeneity in sample analysis

  • Evaluate microenvironment influences on ANAPC1 expression/function

• Advanced experimental design:

  • Implement in vivo models with tissue-specific ANAPC1 modulation

  • Use patient-derived xenografts to maintain tumor heterogeneity

  • Employ single-cell approaches to resolve population heterogeneity

• Integrated data analysis:

  • Correlate ANAPC1 expression with comprehensive molecular profiling

  • Perform multivariate analysis incorporating clinical parameters

  • Apply machine learning approaches to identify contextual patterns

The recent finding of ANAPC1 overexpression in lung squamous cell carcinoma with prognostic implications provides a starting point for systematic investigations across cancer types.

How can researchers effectively test potential therapeutic compounds targeting ANAPC1 in cancer models?

Based on recent findings identifying tenovin-1, carboxyatractyloside, and phycocyanobilin as potential antitumor agents targeting ANAPC1 , researchers can implement a systematic testing approach:

• Compound binding validation:

  • Perform molecular docking simulations to predict binding sites

  • Validate binding using thermal shift assays or surface plasmon resonance

  • Conduct competition assays with known ANAPC1 interactors

• Functional screening cascade:

  • Primary screen: cell viability in ANAPC1-overexpressing cancer lines

  • Secondary screen: impact on APC/C complex formation and activity

  • Tertiary screen: cell cycle progression and mitotic regulation

• Mechanism of action studies:

  • Assess effects on ANAPC1 protein levels and stability

  • Evaluate impact on ANAPC1 subcellular localization

  • Determine effects on ANAPC1 post-translational modifications

• Predictive biomarker development:

  • Correlate drug sensitivity with ANAPC1 expression levels

  • Identify genetic or molecular features predicting response

  • Develop companion diagnostic approaches using ANAPC1 antibodies

• In vivo validation approach:

  • Test efficacy in xenograft models with varying ANAPC1 expression

  • Evaluate pharmacokinetics and biodistribution

  • Implement combination studies with standard-of-care therapies

This systematic approach leverages the recent finding that ANAPC1 knockdown inhibits cancer cell proliferation , suggesting therapeutic potential in targeting this protein.

What methodological approaches can elucidate ANAPC1's role in immune regulation and immunotherapy response?

Recent research indicates ANAPC1 overexpression reduces immune cell infiltration and immunotherapy effectiveness . To further investigate this immune regulatory role:

• Immune profiling in ANAPC1-modulated systems:

  • Flow cytometry to quantify immune cell populations

  • Spatial transcriptomics to assess immune cell distribution

  • Cytokine profiling to identify altered signaling pathways

• Mechanistic investigation:

  • Co-culture systems with ANAPC1-modified cancer cells and immune cells

  • Evaluation of antigen presentation machinery in ANAPC1-high/low cells

  • Assessment of immunomodulatory molecule expression (PD-L1, etc.)

• Clinical correlation studies:

  • IHC analysis of ANAPC1 expression and immune infiltrates in patient samples

  • Correlation of ANAPC1 levels with immunotherapy response metrics

  • Development of composite biomarkers incorporating ANAPC1 and immune signatures

• Therapeutic targeting approaches:

  • Combination strategies targeting ANAPC1 and immune checkpoints

  • Evaluation of immunotherapy sensitization through ANAPC1 inhibition

  • Development of ANAPC1-targeting antibody-drug conjugates

• Translational validation:

  • Prospective analysis of ANAPC1 expression in immunotherapy trials

  • Preclinical models testing ANAPC1-based patient selection strategies

  • Assessment of dynamic ANAPC1 changes during immunotherapy

Understanding ANAPC1's immunomodulatory functions could significantly impact patient selection strategies for cancer immunotherapies and lead to novel combination approaches.