ANAPC4 Antibody

What is ANAPC4 and why is it important in cell cycle research?

ANAPC4 (Anaphase Promoting Complex Subunit 4) is a crucial 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. This large protein complex promotes metaphase-anaphase transition by ubiquitinating specific substrates such as mitotic cyclins and anaphase inhibitors, which are subsequently degraded by the 26S proteasome .

The importance of ANAPC4 in cell cycle research stems from its role in:

• Mediating the formation of 'Lys-11'-linked polyubiquitin chains (primary activity)

• Formation of 'Lys-48'- and 'Lys-63'-linked polyubiquitin chains (secondary activity)

• Controlling the precise timing of cell division events

• Ensuring proper chromosome segregation during mitosis

Researchers investigating cell cycle dysregulation in cancer and other disorders often study ANAPC4 as part of the APC/C complex to understand mechanisms of cellular proliferation.

What are the primary research applications for ANAPC4 antibodies?

ANAPC4 antibodies serve multiple research applications with varying recommended dilutions depending on the specific antibody and application:

When designing experiments, researchers should note that the optimal dilution is antibody-specific and may require optimization. Many publications report successful ANAPC4 detection in human, mouse, and rat samples using these applications .

How does ANAPC4 function within the APC/C complex structure?

ANAPC4 functions as a component of the scaffolding subcomplex platform within the APC/C complex. The APC/C consists of three subcomplexes:

• Scaffolding subcomplex platform: Contains APC1/TSG24, APC4, and APC5

• Catalytic and substrate identification core: Contains APC2, APC10, and RING finger protein APC11

• TPR arm: Contains APC3, APC6, APC7, and APC8, providing binding sites for scaffolding and coactivators (Cdc20 or Cdh1)

Within this architecture, APC4 serves as part of the platform that bridges the catalytic portion with other components. The APC1 subunit functions as a bridge between the catalytic portion and the TPR arm. Together, these components enable the complex to recruit specific substrates for ubiquitination .

Research has demonstrated that APC4 associates with other APC/C components (APC1, APC5, and CDC23) interdependently, such that loss of any one subunit reduces binding between the remaining three , highlighting its structural importance.

What are the recommended sample preparation techniques for detecting ANAPC4 in different tissue types?

Sample preparation for ANAPC4 detection varies by application and tissue type:

For Western Blot applications:

• Human samples: HEK-293 cells, human brain tissue, HeLa cells, and MCF-7 cells have shown positive results

• Mouse samples: NIH/3T3 cells and mouse liver tissue lysate have demonstrated good detection

• Protein extraction should use buffers containing protease inhibitors to prevent degradation

• Expected molecular weight is 92 kDa, though a 63 kDa band has also been observed

For IHC applications:

• Mouse brain tissue has shown positive results with suggested antigen retrieval using TE buffer pH 9.0

• Alternative antigen retrieval may be performed with citrate buffer pH 6.0

• Human thyroid cancer and gastric cancer tissues have shown positive staining at 1:30 dilution

• Paraffin-embedded samples typically yield better results than frozen sections

For optimal results, tissue-specific modifications may be necessary, and researchers should validate the antibody in their specific experimental system.

What controls should be included when using ANAPC4 antibodies in research protocols?

Proper experimental controls are crucial for validating ANAPC4 antibody specificity and ensuring reliable results:

Positive controls:

• Cell lines with known ANAPC4 expression: HEK-293, HeLa, and MCF-7 cells

• Tissues with confirmed expression: human brain tissue, mouse brain tissue

• Recombinant ANAPC4 protein (when available)

Negative controls:

• Primary antibody omission (to detect non-specific binding of secondary antibody)

• Isotype controls (matching the host species and immunoglobulin class)

• Blocking peptide competition assays (using the immunizing peptide)

• ANAPC4-depleted samples via immunoprecipitation or genetic knockdown

Validation controls:

• Different antibody clones targeting distinct epitopes of ANAPC4

• Comparison with gene expression data (RNA levels)

• ANAPC4 knockdown or knockout samples where possible

For advanced applications, synthetic peptide competition assays have been effectively used to validate specificity, as demonstrated with PACO18571 antibody on human thyroid cancer tissue .

How can researchers optimize Western blot protocols for ANAPC4 detection?

Optimizing Western blot protocols for ANAPC4 detection requires careful attention to several parameters:

Sample preparation:

• Use fresh samples when possible

• Include protease inhibitors in lysis buffers

• Determine optimal protein concentration (typically 40-50 μg per lane)

Gel selection and transfer:

• Use 6-8% SDS-PAGE gels for better resolution of the 92 kDa ANAPC4 protein

• For transfer, semi-dry transfer systems work well with appropriate transfer time adjustments

Antibody incubation:

• Primary antibody dilution: Start with 1:1000 and adjust as needed (range: 1:1000-1:6000)

• Secondary antibody: Anti-rabbit IgG for most commercially available ANAPC4 antibodies

• Extended primary antibody incubation (overnight at 4°C) often improves signal quality

Detection systems:

• ECL-based detection systems provide sufficient sensitivity for most applications

• For weaker signals, consider enhanced chemiluminescence substrates

Troubleshooting guidance:

• If detecting multiple bands, check for potential isoforms (63 kDa band has been reported alongside the expected 92 kDa band)

• If no signal appears, consider increasing antibody concentration or protein loading

• High background may require increased washing steps or blocking optimization

How can ANAPC4 antibodies be employed in studying the APC/C complex function during mitosis?

Advanced researchers can employ ANAPC4 antibodies to investigate APC/C complex dynamics during mitosis through several sophisticated approaches:

Immunodepletion studies:
ANAPC4 antibodies can be used to immunodeplete APC/C from cell extracts, creating a system to study mitotic progression in the absence of functional APC/C. Research has shown that ANAPC4 immunoprecipitation co-depletes catalytically active subunits ANAPC2, ANAPC11, and ANAPC10, resulting in reduced UBE2C-dependent ubiquitination .

In vitro ubiquitination assays:
ANAPC4 antibody-conjugated beads can be mixed with cell extracts (1 μg antibody to 166 μg extract ratio) to isolate active APC/C complexes for in vitro ubiquitination studies . This approach allows researchers to:

• Identify novel APC/C substrates

• Study ubiquitination kinetics

• Evaluate effects of regulatory proteins on APC/C activity

Mitotic checkpoint complex (MCC) interaction studies:
ANAPC4 antibodies can help investigate how the MCC (containing MAD2/MAD3, BUB3, and Cdc20) interacts with APC/C to prevent premature anaphase onset, a fundamental aspect of the spindle assembly checkpoint .

E2 enzyme specificity analysis:
By using ANAPC4 antibodies to isolate APC/C complexes, researchers can explore the specificity of different E2 enzymes (UBE2C and UBE2S) in building specific types of ubiquitin chains on APC/C substrates .

What methodological approaches can identify novel ANAPC4-dependent ubiquitination targets?

Identifying novel ANAPC4-dependent ubiquitination targets requires sophisticated methodological approaches:

E2~dID (E2-ubiquitin thioester-driven identification):
This advanced technique has successfully identified APC/C substrates by:

• Performing reactions with UBE2C K119R~bioUBB (a modified E2 enzyme) in anaphase extracts

• Comparing results between normal and ANAPC4-depleted extracts

• Purifying bioUBB-modified substrates using NeutrAvidin beads

• Identifying candidates by mass spectrometry

This approach identified 60 high-confidence hits, with 30 previously reported and 26 uncharacterized potential APC/C substrates .

Quantitative diGly proteomics:
This method combines:

• ANAPC4 depletion using auxin-induced degradation systems

• TMT-labeling of peptides

• diGly-enrichment to identify ubiquitinated peptides

• Mass spectrometry analysis

Using this approach, researchers identified over 18,000 peptides with diGly signatures, with 268 peptides mapping to known mitotic APC/C substrates and 260 peptides corresponding to candidates suggested by E2~dID .

Selection criteria for confident identification:

• ≥2-fold enrichment in ubiquitination reactions compared to controls

• Reduced ubiquitination in ANAPC4-depleted samples

• Presence of APC/C recognition motifs (D-box, KEN-box)

• Temporal correlation with APC/C activity during cell cycle progression

How can researchers distinguish between ANAPC4-specific signals and non-specific antibody binding in complex tissues?

Distinguishing specific ANAPC4 signals from non-specific binding in complex tissues requires rigorous validation approaches:

Peptide competition assays:
Pre-incubate the ANAPC4 antibody with excess immunizing peptide (when available) before application to tissue sections. Specific signals should be significantly reduced or eliminated. The PACO18571 antibody has demonstrated this in human thyroid and gastric cancer tissues .

Multiple antibody validation:
Use antibodies targeting different ANAPC4 epitopes:

• N-terminal targeting antibodies (e.g., Boster A06703)

• Center region targeting antibodies (e.g., AMS.AP17878c-ev)

• C-terminal targeting antibodies

If all show similar staining patterns, specificity is more likely.

Genetic approaches:

• CRISPR/Cas9-mediated depletion: The Zhang lab has designed specific gRNA sequences to target ANAPC4

• RNAi: Use siRNA/shRNA to knockdown ANAPC4 and confirm reduced antibody signal

• Auxin-inducible degron systems: As demonstrated with 3xFlag-Streptavidin-binding-peptide-Venus-ANAPC4, which showed ~75% depletion after 3 hours of 1-naphthaleneacetic acid (NAA) treatment

Advanced imaging techniques:

• Super-resolution microscopy to examine subcellular localization consistent with known ANAPC4 distribution

• Co-localization with other APC/C components (APC2, APC11) as an additional specificity control

• Proximity ligation assays to verify interaction with known binding partners

How should researchers interpret variations in ANAPC4 molecular weight observed in Western blots?

Variations in ANAPC4 molecular weight on Western blots require careful interpretation:

Expected vs. observed molecular weights:

• Calculated molecular weight: 92 kDa

• Commonly observed additional band: 63 kDa

This discrepancy could reflect:

• Isoform detection: ANAPC4 has multiple isoforms. At least two transcript variants encoding different isoforms have been identified . These may migrate differently on SDS-PAGE.

• Post-translational modifications: Phosphorylation, ubiquitination, or other modifications can alter migration patterns.

• Proteolytic processing: Partial degradation during sample preparation might generate lower molecular weight fragments.

• Splice variants: Alternative splicing could produce truncated protein versions.

To determine which explanation applies:

• Compare results using antibodies targeting different ANAPC4 epitopes

• Employ phosphatase treatment to identify phosphorylation-dependent mobility shifts

• Confirm with mass spectrometry to identify the exact nature of the observed proteins

• Check databases for reported isoforms and their expected molecular weights

What are common pitfalls in ANAPC4 immunohistochemistry and how can they be addressed?

Common pitfalls in ANAPC4 immunohistochemistry include:

High background staining:

• Cause: Insufficient blocking, excessive antibody concentration, or cross-reactivity

• Solution: Optimize blocking (increase BSA/serum concentration), titrate antibody dilution (start with 1:50-1:500 range) , and use longer/more stringent wash steps

Weak or absent signal:

• Cause: Inadequate antigen retrieval, epitope masking, or insufficient antibody concentration

• Solution: Try alternative antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0 as suggested for 14129-1-AP antibody) . Consider testing different antibody clones.

Non-specific nuclear staining:

• Cause: ANAPC4 is primarily nuclear, but excessive nuclear staining may indicate non-specific binding

• Solution: Validate with peptide competition controls and compare with known expression patterns

Variable staining between tissue regions:

• Cause: Fixation inconsistencies or processing artifacts

• Solution: Standardize fixation protocols and processing times; consider automated IHC platforms

Optimization recommendations:
For human tissues: Test both 1:30 dilution (as used with PACO18571) and 1:50-1:500 range (as recommended for 14129-1-AP)
For mouse brain tissue: Use TE buffer pH 9.0 for antigen retrieval

How can discrepancies in ANAPC4 detection between different antibodies be reconciled?

Discrepancies in ANAPC4 detection between different antibodies may arise from several factors:

Epitope targeting differences:
Different antibodies target distinct regions of ANAPC4:

• N-terminal antibodies (e.g., Boster A06703 targeting residues 40-90)

• Center region antibodies (e.g., AMS.AP17878c-ev)

• C-terminal antibodies

If discrepancies appear, consider:

• Isoform specificity: Some antibodies may detect only specific isoforms

• Epitope accessibility: In certain applications, some epitopes may be masked by protein folding or interactions

• Post-translational modifications: Modifications near epitopes may affect antibody binding

Reconciliation strategies:

• Orthogonal validation: Confirm results using non-antibody-based methods (e.g., mass spectrometry, RNA expression)

• Combined approach: Use multiple antibodies targeting different epitopes and look for consensus results

• Genetic validation: Use CRISPR/Cas9 knockdown with gRNAs designed to target ANAPC4 and verify signal reduction with all antibodies

• Specific applications: Determine which antibody performs best for each application type (WB, IHC, IP, etc.)

Documentation practices:
Thoroughly document antibody details in publications:

• Catalog number

• Lot number

• Host species

• Clonality

• Immunogen information

This information facilitates result comparison and experimental reproducibility across research groups.

How are ANAPC4 antibodies advancing our understanding of APC/C structure and function?

ANAPC4 antibodies have significantly advanced our understanding of APC/C structure and function in several key ways:

Complex assembly mechanisms:
Immunoprecipitation studies using ANAPC4 antibodies have revealed that APC4 associates with other APC/C components (APC1, APC5, and CDC23) interdependently, with the loss of any one subunit reducing binding between the remaining three . This has enhanced our understanding of APC/C assembly.

Subcellular localization:
Immunofluorescence using ANAPC4 antibodies has helped define the primarily nuclear localization of APC/C components, particularly at the nuclear periphery . This spatial information provides context for APC/C function.

Structural insights:
ANAPC4 antibodies have facilitated purification of intact APC/C complexes for structural studies, revealing that APC4 contains:

• An N-terminal WD40 domain

• A C-terminal long domain
  These domains are critical for APC/C scaffolding .

E2 enzyme recruitment mechanisms:
Studies using ANAPC4 immunodepletion have clarified how the APC/C complex sequentially recruits different E2 enzymes (UBE2C and UBE2S) to build specific ubiquitin chains . This has expanded our understanding of the complex mechanisms underlying APC/C-mediated ubiquitination.

Cell cycle regulation:
Investigators have used ANAPC4 antibodies to study how the mitotic checkpoint complex (MCC) inhibits APC/C activity during the spindle assembly checkpoint, preventing premature anaphase onset .

What emerging technologies are enhancing ANAPC4 antibody applications in research?

Several emerging technologies are enhancing ANAPC4 antibody applications:

Auxin-inducible degron systems:
Researchers have developed systems using mAID-vhhGFP to target proteins fused to GFP or GFP-like proteins. This approach has successfully depleted endogenous 3xFlag-Streptavidin-binding-peptide-Venus-ANAPC4 by approximately 75% after three hours of 1-naphthaleneacetic acid (NAA) treatment . This allows:

• Rapid and reversible ANAPC4 depletion

• Temporal control of APC/C function

• Creation of APC/C-deficient cells for substrate identification

CRISPR/Cas9 genome editing:
The Zhang laboratory has designed specific gRNA sequences uniquely targeting the ANAPC4 gene with minimal off-target effects . These tools enable:

• Creation of ANAPC4 knockout cell lines

• Generation of epitope-tagged endogenous ANAPC4

• Introduction of specific mutations to study structure-function relationships

Quantitative diGly proteomics:
This advanced approach combines ANAPC4 depletion with TMT-labeling and diGly-enrichment to comprehensively identify APC/C substrates . The technique has identified:

• Over 18,000 peptides with diGly signatures

• 268 peptides mapping to known mitotic APC/C substrates

• 260 peptides from potential novel substrates

E2~dID (E2-ubiquitin thioester-driven identification):
This methodology uses modified E2 enzymes and biotin-labeled ubiquitin to identify APC/C substrates in the presence and absence of ANAPC4 . This approach has identified 60 high-confidence APC/C substrates, including 26 previously uncharacterized candidates.

What role might ANAPC4 antibodies play in elucidating links between APC/C dysfunction and disease processes?

ANAPC4 antibodies are poised to make significant contributions to understanding the links between APC/C dysfunction and disease processes:

Cancer research applications:
APC/C dysregulation is associated with genomic instability and cancer development. ANAPC4 antibodies provide valuable tools for:

• Analyzing ANAPC4 expression in different cancer types using tissue microarrays

• Identifying altered APC/C complex formation in tumor samples

• Studying mechanisms of mitotic checkpoint defects in cancer cells

Current evidence showing successful application includes immunohistochemical analysis of human thyroid and gastric cancer tissues using ANAPC4 antibodies (PACO18571) . These antibodies "could lead to significant advancements in cancer research and drug discovery" .

Neurodegenerative disease investigations:
Emerging evidence suggests potential roles for APC/C in neurodegenerative disorders. ANAPC4 antibodies can help:

• Examine APC/C expression in neuronal tissues

• Investigate non-canonical functions of APC/C in post-mitotic neurons

• Study potential links to protein aggregation mechanisms

ANAPC4 antibodies have already demonstrated successful staining in human and mouse brain tissues , providing a foundation for these investigations.

Therapeutic development:
ANAPC4 antibodies are instrumental in validating the effects of APC/C-targeting therapies:

• Evaluating target engagement of potential APC/C inhibitors

• Assessing changes in complex formation in response to treatment

• Monitoring compensatory mechanisms that might affect therapeutic efficacy