ANAPC2 Antibody

What is ANAPC2 and what is its biological function?

ANAPC2 is a core subunit 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. Together with ANAPC11 (a RING-H2 protein), it constitutes the catalytic component of the APC/C complex . The primary function of this complex is mediating ubiquitination and subsequent degradation of target proteins, mainly through the formation of 'Lys-11'-linked polyubiquitin chains, and to a lesser extent, 'Lys-48' and 'Lys-63'-linked chains .

ANAPC2 specifically forms a heterodimeric complex with either Ubc4 or UbcH10 and APC11, which is responsible for ubiquitinating key cell cycle proteins including human securin and cyclin B1 . The C-terminal cullin homology domain of ANAPC2 binds both APC11 and UBE2C, enabling the complex to assemble branched 'Lys-11'-/'Lys-48'-linked ubiquitin chains on target proteins .

Beyond mitotic regulation, the CDC20-APC/C complex (which includes ANAPC2) positively regulates the formation of synaptic vesicle clustering at active zones in postmitotic neurons, demonstrating that ANAPC2 has functions beyond cell cycle control .

How can I determine if an ANAPC2 antibody will recognize my species of interest?

When selecting an ANAPC2 antibody for your research, species reactivity is a critical consideration. Commercial antibodies are typically tested against specific species, with varying degrees of validation. For example, antibody suppliers like Abcam categorize their products based on testing status:

• Fully tested and confirmed working: These antibodies have been tested in the specific species and application of interest and are covered by product guarantees .

• Expected to work: These antibodies have not been specifically tested for your species/application combination but are expected to work based on the supplier's experience with similar combinations .

• Predicted to work based on homology: These antibodies have not been tested for your specific combination but are predicted to work based on sequence homology between species .

• Not recommended: These combinations are not expected to work and are not covered by product guarantees .

To determine cross-reactivity potential beyond what suppliers list, researchers should consider:

• Conducting sequence alignment analysis of the immunogen region across species

• Performing preliminary validation experiments with positive and negative controls

• Consulting published literature where the antibody has been used in your species of interest

What are the optimal applications for detecting ANAPC2 in experimental settings?

Based on validated antibody products, ANAPC2 can be reliably detected using several experimental techniques. The optimal applications depend on the specific research question:

For complex tissues like bone marrow samples, the bi-color IHC staining approach has proven effective. This method involves first incubating with anti-CD34 antibody (for identifying hematopoietic stem cells), followed by anti-ANAPC2 antibody detection using a separate chromogen . This sequential approach allows for colocalization studies of ANAPC2 with other markers of interest.

How should I optimize immunohistochemistry protocols for ANAPC2 detection in tissue samples?

For effective ANAPC2 detection in tissue samples using immunohistochemistry, consider this optimized protocol based on successful research applications:

• Antigen retrieval: Use citrate buffer (pH 6.0) at 98°C for 12 minutes .

• Blocking steps:

  • Block endogenous peroxidase activity with 3% H₂O₂ at 37°C for 10 minutes

  • Block non-specific binding sites with 5% normal goat serum at room temperature for 15 minutes

• Primary antibody incubation: Apply anti-ANAPC2 antibody (1:200 dilution in PBS pH 7.4 containing 1% BSA and 2% FCS) and incubate at 4°C overnight .

• Detection system: Use HRP-Polymer anti-Rabbit IHC Kit followed by an appropriate chromogen such as DAB or HighDef® yellow IHC chromogen .

• Counterstaining: Nuclear fast red provides good contrast for visualizing positive ANAPC2 staining .

• Controls: Include both positive controls (tissues known to express ANAPC2) and negative controls (omit primary antibody) in each experiment.

• Analysis: Have at least two investigators examine the staining results to ensure reliable interpretation .

For dual-staining approaches, sequential application of antibodies with distinct chromogens allows visualization of ANAPC2 alongside other proteins of interest, as demonstrated in studies examining ANAPC2 expression in CD34+ hematopoietic stem cells .

What are the critical considerations for quantifying ANAPC2 mRNA levels?

Accurate quantification of ANAPC2 mRNA levels requires careful attention to several methodological aspects:

• RNA extraction and quality control:

  • Use TRIzol® Reagent or equivalent for total RNA purification from cells or tissues

  • Assess RNA integrity using gel electrophoresis or Bioanalyzer before proceeding

• Primer design for qPCR:

  • Target specific regions of ANAPC2 mRNA, such as those amplified by validated primers:

    • Forward primer: 5'-TATGTTGCGCGGAGTCTTGTT-3'

    • Reverse primer: 5'-GAAGCACCCATACAGACGCTG-3'

  • Test primer efficiency using standard curves

  • Ensure primers span exon-exon junctions to avoid genomic DNA amplification

• Reference gene selection:

  • Use stable reference genes like β-actin for normalization

  • Consider validating multiple reference genes for your specific experimental conditions

• Data analysis:

  • Apply the comparative Ct (2^-ΔΔCt) method for relative quantification

  • Express results as normalized to reference genes and relative to control samples

  • Present data as mean ± SEM based on at least three independent experiments with triplicate samples

• Validation:

  • Confirm major findings with protein-level measurements (Western blot)

  • Consider alternative approaches such as RNA-seq for more comprehensive analysis

How does ANAPC2 deletion affect hematopoietic stem and progenitor cells?

Research using conditional knockout mouse models has revealed critical roles for ANAPC2 in hematopoiesis. When Anapc2 is deleted in hematopoietic cells using a Cre-LoxP system, it leads to:

• Rapid bone marrow failure: Conditional Anapc2 knockout mice develop fatal bone marrow failure within 7 days after knockout induction .

• Sharp decline in hematopoietic stem and progenitor cells (HSPCs):

  • Flow cytometry analysis shows that LSK (Lin⁻Sca-1⁺c-Kit⁺) cells drop precipitously at day 2 post-induction and become nearly undetectable by day 3

  • Immunofluorescence staining confirms significant reduction of c-Kit+ cells by day 5

• Loss of colony-forming capacity: LSK cells from Anapc2-deleted mice can hardly generate any colonies in colony formation cell (CFC) assays, indicating cell-intrinsic defects .

• Increased apoptosis: Annexin V and PI double staining reveals a significant increase in Annexin V+ LSK cells by day 3 after Anapc2 deletion, demonstrating that these cells undergo programmed cell death .

• Loss of dormant HSPCs: BrdU label-retaining cell assays show that dormant hematopoietic stem cells are rapidly lost following Anapc2 deletion, suggesting a shift from quiescence to mitosis followed by apoptosis .

These findings demonstrate that ANAPC2, and by extension the APC/C complex, is essential for the maintenance and survival of hematopoietic stem and progenitor cells, particularly for preserving their quiescent state.

What is the relationship between ANAPC2 expression and aplastic anemia?

Research investigating ANAPC2 expression in patients with aplastic anemia (AA) has revealed intriguing connections that may have pathophysiological significance:

• Reduced CD34+ cells: AA patients show markedly decreased numbers of CD34+ hematopoietic stem/progenitor cells in bone marrow compared to normal controls .

• Absent ANAPC2 expression: Importantly, the residual CD34+ cells in AA bone marrow samples demonstrate undetectable levels of ANAPC2 expression, while CD34+ cells from normal controls show clear ANAPC2 expression .

• Potential mechanism: The absence of ANAPC2 in human CD34+ cells in AA parallels the findings from mouse models where Anapc2 deletion causes rapid bone marrow failure . This suggests that APC/C deficiency might contribute to the pathogenesis of AA by compromising HSPC maintenance.

• Diagnostic implications: CD34/ANAPC2 bi-color immunohistochemistry staining could potentially serve as a diagnostic tool for evaluating AA cases, though larger clinical studies would be needed to validate this approach .

These observations suggest a potential role for ANAPC2 deficiency in the pathogenesis of aplastic anemia, a bone marrow failure disease characterized by pancytopenia and hypocellular bone marrow. The similarities between the phenotypes observed in Anapc2 knockout mice and human AA patients provide a compelling direction for further investigation into APC/C dysfunction as a contributing factor to bone marrow failure syndromes.

How does the ANAPC2-containing APC/C complex regulate ubiquitination of target proteins?

The ANAPC2-containing APC/C complex employs sophisticated mechanisms to regulate protein degradation through ubiquitination:

• Catalytic core formation: ANAPC2 partners with ANAPC11 to form the catalytic core of the APC/C complex. The C-terminal cullin homology domain of ANAPC2 binds both ANAPC11 and the E2 ubiquitin-conjugating enzyme UBE2C .

• Ubiquitin chain diversity: The APC/C complex predominantly catalyzes the formation of 'Lys-11'-linked polyubiquitin chains, though it also mediates 'Lys-48' and 'Lys-63'-linked chains to a lesser extent .

• Branched chain assembly: Recent research has demonstrated that the APC/C complex catalyzes the assembly of branched 'Lys-11'/'Lys-48'-linked ubiquitin chains on target proteins, which may enhance recognition by the proteasome .

• Co-activator dependence: The activity of the APC/C complex is regulated by co-activators such as CDC20 and CDH1, which recognize specific substrates through degron motifs like the D-box and KEN-box .

• Key substrates: In cell cycle regulation, APC/C targets critical proteins including:

  • Securin, allowing sister chromatid separation

  • Cyclins, enabling exit from mitosis

  • Other regulatory proteins that must be degraded for proper cell division

• Non-mitotic functions: In neurons, CDC20-APC/C regulates presynaptic differentiation through the degradation of NEUROD2, demonstrating substrate diversity beyond cell cycle regulators .

Understanding these mechanisms has significant implications for developing targeted approaches to modulate APC/C activity in diseases where cell cycle dysregulation plays a role.

How can I validate the specificity of ANAPC2 antibodies for my research?

Ensuring antibody specificity is crucial for generating reliable data. Here are comprehensive validation approaches for ANAPC2 antibodies:

• Positive and negative controls:

  • Positive controls: Use cell lines or tissues known to express ANAPC2 (based on literature)

  • Negative controls: Include samples where primary antibody is omitted

  • Genetic controls: If available, use ANAPC2 knockout/knockdown samples as the gold standard for specificity

• Multiple detection methods:

  • Compare results across different techniques (WB, IHC, ICC) using the same antibody

  • Confirm key findings using at least two different antibodies targeting distinct epitopes of ANAPC2

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide

  • A specific antibody will show reduced or eliminated signal when blocked with its target peptide

• Molecular weight verification:

  • For Western blotting, confirm that the detected band matches the expected molecular weight of ANAPC2

  • Watch for potential post-translational modifications that may alter apparent molecular weight

• Immunoprecipitation followed by mass spectrometry:

  • Use the antibody for immunoprecipitation and verify pulled-down proteins by mass spectrometry

  • This can identify both the target protein and potential cross-reactive proteins

• Multiple investigator review:

  • Have at least two trained investigators examine and interpret staining results independently

  • This reduces subjective bias in qualitative assessments

• Publication records:

  • Review literature where the antibody has been used, particularly papers with rigorous validation

  • Note any reported limitations or specific conditions for optimal performance

What are the optimal methods for analyzing ANAPC2 in hematopoietic stem and progenitor cells?

Analysis of ANAPC2 in hematopoietic stem and progenitor cells (HSPCs) requires specialized approaches due to their rarity and unique characteristics:

• Flow cytometry:

  • Use lineage depletion to enrich for HSPCs before analysis

  • Identify LSK (Lin⁻Sca-1⁺c-Kit⁺) cells using fluorochrome-conjugated antibodies against lineage markers (CD11b, Gr-1, B220, CD3, TER-119), Sca-1, and c-Kit

  • For apoptosis assessment, combine with Annexin V and PI staining

  • Analyze using a high-sensitivity flow cytometer like LSRFortessa and sort using FACSAriaII or equivalent

• Immunohistochemistry/Immunofluorescence:

  • For human samples, use CD34 as a marker for HSPCs

  • Implement bi-color IHC staining with CD34 and ANAPC2 antibodies using different chromogens to assess colocalization

  • For mouse samples, c-Kit staining can help identify HSPCs in bone marrow sections

• Functional assays:

  • Colony-formation cell (CFC) assay in MethoCult GF M3434 medium to assess HSPC function

  • Plate 600-700 sorted LSK cells and evaluate colony formation after 14 days

• Gene expression analysis:

  • Use qPCR with validated primers to quantify ANAPC2 mRNA levels

  • Consider single-cell approaches for heterogeneous HSPC populations

• Spatiotemporal considerations:

  • Note that HSPCs are enriched in the metaphysis of long bones

  • When analyzing bone marrow sections, assess both metaphysis and diaphysis regions to capture the distribution of HSPCs and potential changes following experimental manipulation

• Data presentation:

  • Report both percentage and absolute numbers of HSPCs

  • Present data as mean ± SEM from multiple independent experiments

What controls should be included in ANAPC2 knockout studies?

When conducting ANAPC2 knockout studies, proper controls are essential for generating interpretable and reliable results:

• Genotype verification controls:

  • PCR verification of genotype using primers specific to the floxed allele and Cre recombinase

  • Example primers for Anapc2: F1-(5′-GCGACAATTATTGCCTCCGATGACTGCGAC-3′), R1-(5′-TGGAGAACCCACAACACACATCTGTCCCTTACC-3′)

  • Example primers for Cre: F-(5′-GCGGTCTGGCAGTAAAACTATC-3′), R-(5′-AGCAATCCCCAGAAATGCCAG-3′)

• Knockout validation controls:

  • Confirmation of gene deletion at genomic DNA level

  • Verification of mRNA reduction by qPCR

  • Demonstration of protein absence by Western blot

• Temporal controls:

  • For inducible systems (e.g., Mx1-Cre), include time-course experiments to track phenotypic progression

  • Monitor changes at multiple timepoints (e.g., day 1, 3, 5, 7) after knockout induction

• Wild-type controls:

  • Use littermates without Cre recombinase (e.g., Anapc2ᶠˡᵒˣ/ᶠˡᵒˣ without Mx1-Cre) as the proper control group

  • This controls for genetic background effects and potential toxicity of inducers like pIpC

• Induction controls:

  • For inducible systems, include control animals that receive the inducer (e.g., pIpC) but lack either the floxed allele or the Cre recombinase

  • This controls for potential side effects of the induction method

• Rescue experiments:

  • When possible, perform rescue experiments by reintroducing wild-type ANAPC2 to confirm that observed phenotypes are specifically due to ANAPC2 loss

• Cell-specific controls:

  • For tissue-specific knockout studies, include analysis of ANAPC2 expression in non-targeted tissues to confirm specificity of the deletion

By incorporating these controls, researchers can rigorously attribute observed phenotypes to ANAPC2 deletion rather than experimental artifacts or off-target effects.

What are emerging areas of investigation regarding ANAPC2 function beyond cell cycle regulation?

While ANAPC2's role in cell cycle regulation through the APC/C complex is well-established, research is uncovering additional functions that merit further investigation:

• Neurological functions: The CDC20-APC/C complex, which includes ANAPC2, positively regulates synaptic vesicle clustering at active zones and drives presynaptic differentiation through NEUROD2 degradation . This suggests broader roles in neuronal development and function that remain to be fully characterized.

• Stem cell biology: The essential role of ANAPC2 in maintaining hematopoietic stem cell quiescence raises questions about whether similar mechanisms operate in other stem cell populations, such as neural stem cells, mesenchymal stem cells, or intestinal stem cells.

• Disease associations: The correlation between ANAPC2 deficiency and aplastic anemia suggests potential roles in other hematological disorders or diseases characterized by dysregulated cell cycling. Investigating ANAPC2 expression and function in conditions like myelodysplastic syndromes or leukemias could yield valuable insights.

• Therapeutic targeting: Given ANAPC2's essential role in the catalytic core of APC/C, developing small molecules that modulate this complex could provide new therapeutic approaches for diseases with aberrant cell cycle regulation.

• Interactome mapping: Comprehensive identification of ANAPC2 binding partners beyond the core APC/C complex could reveal unexpected functions and regulatory mechanisms.

Future research using advanced techniques such as CRISPR-Cas9 genome editing, single-cell analysis, and in vivo imaging will likely uncover additional roles for ANAPC2 in normal physiology and disease states.

How might studying ANAPC2 contribute to our understanding of bone marrow failure diseases?

The discovery of ANAPC2's critical role in hematopoiesis and its potential connection to aplastic anemia opens several promising research avenues:

• Diagnostic biomarker development: The observation that CD34+ cells in aplastic anemia patients lack detectable ANAPC2 expression suggests that ANAPC2 immunostaining could be developed as a diagnostic or prognostic biomarker for bone marrow failure syndromes.

• Pathogenic mechanism elucidation: Further investigation of how ANAPC2 deficiency leads to hematopoietic stem cell depletion could reveal fundamental mechanisms underlying bone marrow failure diseases. Specifically, understanding the transition from quiescence to mitosis followed by apoptosis observed in Anapc2-deleted HSPCs may provide insights into disease progression.

• Genetic screening: Screening for mutations or polymorphisms in ANAPC2 or other APC/C components in patients with bone marrow failure could identify genetic risk factors or subtypes of disease.

• Therapeutic target identification: Understanding the downstream effects of ANAPC2 deficiency, particularly regarding cell cycle regulators like Skp2, P27, Cdk2, and Cyclin E1 , could reveal potential therapeutic targets for preserving or restoring hematopoietic stem cell function.

• Drug development: Compounds that modulate APC/C activity or compensate for ANAPC2 deficiency could potentially be developed as treatments for certain forms of bone marrow failure.

• Modeling disease in vitro: Patient-derived induced pluripotent stem cells (iPSCs) with ANAPC2 knockdown or knockout could provide valuable disease models for studying bone marrow failure mechanisms and testing therapeutic interventions.

These research directions could not only advance our understanding of ANAPC2 biology but also contribute to improved diagnosis and treatment of challenging hematological disorders.