APC2 Antibody

What is APC2 and what cellular functions does it perform?

APC2 (also known as APCL) is a protein that plays dual roles in cellular function. It stabilizes microtubules and regulates actin fiber dynamics through the activation of Rho family GTPases . Additionally, APC2 functions in Wnt signaling by promoting the rapid degradation of β-catenin (CTNNB1) . The protein has a calculated molecular weight of 94 kDa, though it's typically observed at approximately 97 kDa in experimental conditions . APC2 is primarily localized at the cell cortex and in the cytoplasm, with its cortical localization being particularly important for its actin organization functions but dispensable for Wnt signaling regulation .

What applications can APC2 antibodies be used for in research?

APC2 antibodies have been validated for multiple research applications:

What is the expected molecular weight of APC2 in Western blots?

The calculated molecular weight of APC2 is 94 kDa (822 amino acids), but the observed molecular weight in experimental contexts is typically 97 kDa . This slight discrepancy between calculated and observed weights is common for many proteins and may be due to post-translational modifications. When performing Western blot analysis, researchers should look for a band at approximately 100 kDa . If multiple bands are observed, validation experiments such as knockdown/knockout controls should be performed to confirm specificity.

How should I optimize antibody dilution for Western blot detection of APC2?

For optimal Western blot results with APC2 antibodies, begin with the manufacturer's recommended dilution range (1:500-1:3000 for Proteintech antibody or 1:1000 for Cell Signaling antibody) . Start with a mid-range dilution and adjust based on signal strength and background. Consider these optimization steps:

• Test multiple dilutions in parallel on the same membrane if possible

• Include positive controls with known APC2 expression (e.g., HeLa, Jurkat, or MCF-7 cells)

• Use freshly prepared buffers and blocking solutions

• Optimize exposure time to prevent oversaturation or weak signals

• If signal is weak, consider longer primary antibody incubation (overnight at 4°C)

• If background is high, increase washing duration/frequency or adjust blocking conditions

Remember that different detection methods (chemiluminescence, fluorescence) may require different antibody concentrations for optimal results.

What sample preparation techniques maximize APC2 detection in Western blots?

For effective APC2 detection in Western blot applications:

• Use validated cell lines with known APC2 expression (HeLa, Jurkat, or MCF-7 cells are recommended)

• Prepare lysates using complete protease inhibitor cocktails to prevent degradation

• Include phosphatase inhibitors if studying phosphorylation status

• Perform protein quantification and load equal amounts (typically 20-40 μg) per lane

• Use fresh samples when possible or store aliquots at -80°C to avoid freeze-thaw cycles

• Denature samples completely (95°C for 5 minutes) prior to loading

• Use appropriate percentage gels (8-10% typically works well for a 97 kDa protein)

For challenging samples, consider enrichment techniques like immunoprecipitation prior to Western blot analysis to increase sensitivity .

How does APC2 localization affect its role in actin organization versus Wnt signaling?

Research has demonstrated a fascinating functional dichotomy in APC2's cellular roles based on its localization. The cortical localization of APC2 has been found to be essential for its role in organizing actin but, surprisingly, dispensable for its function in regulating Wnt signaling .

Studies in Drosophila S2 cells and embryos have revealed that both the Armadillo repeats and a novel C-terminal domain (C30) are necessary for the cortical localization of APC2, with neither domain alone being sufficient . The Armadillo repeats also mediate self-association of APC2 molecules, potentially contributing to its functional capabilities at the cortex .

When designing experiments to study these distinct functions:

• Consider using domain-specific mutants (particularly Armadillo repeats or C30 deletion constructs) to differentially affect localization

• Use cytoskeletal disrupting agents (e.g., Cytochalasin D) when studying localization dependencies, as actin is required to maintain cortical enrichment of APC2

• Incorporate both Wnt signaling readouts and actin organization assays in the same experimental system to directly compare effects

This domain-specific approach allows researchers to dissect the molecular mechanisms underlying APC2's dual functionality in cellular processes.

What fixation and staining protocols best preserve APC2 cortical localization for immunofluorescence?

For optimal visualization of APC2's cortical localization in immunofluorescence applications:

• Fixation method:

  • Use 4% paraformaldehyde for 15-20 minutes at room temperature

  • Avoid methanol fixation which can disrupt cytoskeletal structures

  • For simultaneous visualization of actin structures, use fresh fixative solutions

• Permeabilization:

  • Gentle permeabilization with 0.1-0.2% Triton X-100 for 5-10 minutes

  • Alternative: 0.05% saponin may better preserve membrane-associated proteins

• Staining protocol:

  • Use APC2 antibody at dilution 1:50-1:500 as recommended

  • Include F-actin counterstaining (phalloidin) to visualize cortical co-localization

  • Include DAPI for nuclear visualization to distinguish cytoplasmic from nuclear signals

• Special considerations:

  • For embryonic tissues, adjust fixation time based on tissue permeability

  • When co-staining with other cortical markers, carefully select secondary antibodies to avoid cross-reactivity

  • Consider antigen retrieval with TE buffer pH 9.0 for tissue sections

Remember that proper control of temperature during staining procedures is crucial for maintaining the integrity of cortical structures. MCF-7 cells have been validated as a positive control for immunofluorescence detection of APC2 .

How can I design experiments to distinguish between APC2's roles in Wnt signaling versus cytoskeletal regulation?

To effectively distinguish between APC2's dual functions:

• Domain-specific approach:

  • Utilize constructs lacking the C-terminal localization domain (C30), which affects cytoskeletal function but not Wnt signaling

  • Compare with full-length APC2 and constructs lacking Armadillo repeats

• Functional readouts:

  • For Wnt signaling: measure β-catenin levels, TOPFlash reporter activity, and expression of Wnt target genes

  • For cytoskeletal function: examine actin organization, particularly pseudocleavage furrows in Drosophila embryos or cortical actin networks in cultured cells

• Pharmacological separation:

  • Use cytoskeletal disruptors (Cytochalasin D) that displace APC2 from the cortex

  • Compare with Wnt pathway modulators to identify differential effects

• Cell type selection:

  • Use S2 cells for basic localization studies as they recapitulate the cortical enrichment observed in embryos

  • Include both Wnt-responsive and non-responsive cell types in parallel experiments

• Microscopy and biochemical analysis combination:

  • Couple high-resolution imaging of APC2 localization with biochemical fractionation

  • Correlate changes in localization with functional outcomes in both pathways

This multi-faceted approach allows for precise dissection of the mechanistic differences between APC2's cytoskeletal and signaling functions.

What are the most common causes of non-specific bands in APC2 Western blots?

When encountering non-specific bands in APC2 Western blots, consider these potential causes and solutions:

• Antibody concentration too high:

  • Dilute primary antibody further within recommended range (1:500-1:3000)

  • Reduce secondary antibody concentration if background remains high

• Cross-reactivity with related proteins:

  • APC family members share sequence homology; validate specificity using knockout controls

  • Use computational analysis to identify potential cross-reactive proteins based on epitope sequence

• Sample degradation:

  • Add fresh protease inhibitors to lysis buffer

  • Maintain samples at appropriate temperature during preparation

  • Run samples immediately after preparation or store properly at -80°C

• Incomplete blocking:

  • Increase blocking time or concentration (typically 5% non-fat dry milk or BSA)

  • Consider alternative blocking agents if background persists

• Insufficient washing:

  • Increase number and duration of wash steps

  • Use gentle agitation during washes

For validation, compare patterns across different cell lines with known APC2 expression (HeLa, Jurkat, MCF-7) and consider using alternative APC2 antibodies raised against different epitopes to confirm band specificity.

How should I interpret contradictory results when studying APC2 in different cell lines?

When facing contradictory results across cell lines:

• Consider cell-specific expression levels:

  • Verify APC2 expression levels in each cell line via qPCR prior to protein studies

  • Compare with published transcriptome databases to confirm expected expression

• Evaluate post-translational modifications:

  • Different cell types may exhibit various modifications affecting antibody recognition

  • Consider phosphatase treatment of samples to eliminate phosphorylation-dependent differences

• Analyze pathway context:

  • The Wnt pathway status differs among cell lines, affecting APC2 function and localization

  • Measure β-catenin levels to determine baseline pathway activity

• Examine subcellular localization:

  • APC2 functions differently based on localization; perform fractionation studies

  • Compare cortical vs. cytoplasmic distribution using immunofluorescence

• Consider genetic background:

  • Check for mutations in APC2 or related pathway components

  • Sequence verification may be necessary in cell lines with unexpected results

When publishing such data, present results from multiple cell lines with appropriate controls and discuss cell-specific differences as potentially biologically meaningful rather than experimental artifacts.

What buffer conditions optimize APC2 antibody performance in immunoprecipitation?

For successful APC2 immunoprecipitation experiments:

Recommended protocol adjustments:

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Pre-clear lysates with protein A/G beads to reduce background

• Incubate antibody-lysate mixture overnight at 4°C with gentle rotation

• Use MCF-7 cells as positive control for IP experiments

• For weaker interactions, consider crosslinking prior to lysis or milder detergents

These conditions have been validated for the Proteintech antibody (13559-1-AP) and can be adapted for other APC2 antibodies with appropriate optimization .

How can I design experiments to study APC2 interactions with Wnt signaling versus cytoskeletal partners?

To effectively differentiate between APC2's interaction networks:

• Compartment-specific immunoprecipitation:

  • Perform fractionation to separate cortical (cytoskeleton-associated) from cytoplasmic pools

  • Conduct parallel IP experiments on each fraction to identify location-specific partners

• Domain-targeted approach:

  • Use deletion constructs lacking either Armadillo repeats or C30 domain

  • Compare interactome differences between these constructs and full-length APC2

• Stimulus-dependent analysis:

  • Compare APC2 interaction partners with/without Wnt pathway activation

  • Similarly, compare before/after cytoskeletal disruption with agents like Cytochalasin D

• Proximity labeling techniques:

  • Consider BioID or APEX2 fusions to APC2 for in vivo proximity labeling

  • Create domain-specific fusions to identify spatial interaction networks

• Validation strategies:

  • Confirm key interactions with reciprocal IPs

  • Use super-resolution microscopy to verify co-localization of interaction partners

  • Perform functional assays (e.g., TOPFlash for Wnt; actin organization assays for cytoskeletal function)

This systematic approach allows for comprehensive mapping of the dual interactomes of APC2, providing insight into how it coordinates its distinct cellular functions.

How can I validate the specificity of my APC2 antibody in knockout/knockdown models?

Rigorous validation of APC2 antibodies is crucial for experimental reliability:

• Genetic knockdown approaches:

  • Implement siRNA or shRNA targeting APC2 in appropriate cell lines

  • Confirm knockdown efficiency at mRNA level via qPCR before protein analysis

  • Published knockdown studies using these antibodies can guide experimental design

• CRISPR/Cas9 knockout validation:

  • Generate complete knockout cell lines using CRISPR/Cas9

  • Verify genomic modification by sequencing

  • Western blot should show complete absence of the 97 kDa APC2 band in knockout lines

• Overexpression controls:

  • Complement knockdown/knockout with APC2 overexpression

  • Use tagged versions (mCherry-APC2 or EGFP-APC2) to distinguish from endogenous protein

  • Verify that antibody detects both endogenous and overexpressed protein

• Cross-validation strategies:

  • Compare results from multiple antibodies targeting different APC2 epitopes

  • Include positive control samples with known APC2 expression (HeLa, Jurkat, MCF-7 cells)

• Application-specific validation:

  • For IHC, include appropriate tissue controls (human breast cancer tissue has been validated)

  • For IF, verify subcellular localization patterns against published data

These validation approaches ensure that observed results are due to specific detection of APC2 rather than antibody cross-reactivity or technical artifacts.

What positive and negative control samples should be included when working with APC2 antibodies?

For robust experimental design with APC2 antibodies:

Positive controls:

• Cell lines with confirmed APC2 expression:

  • HeLa cells (human cervical cancer)

  • Jurkat cells (human T lymphocyte)

  • MCF-7 cells (human breast cancer)

• Tissue samples with validated expression:

  • Human breast cancer tissue (for IHC)

  • Human brain cortex (for IHC)

  • Human small intestine (for IHC)

Negative controls:

• Technical negative controls:

  • Primary antibody omission control

  • Isotype control (rabbit IgG at equivalent concentration)

  • Secondary antibody-only control

• Biological negative controls:

  • APC2 knockout or knockdown samples

  • Cell lines with verified low/no APC2 expression (should be validated)

Application-specific controls:

• For Western blotting:

  • Molecular weight ladder to confirm correct band size (97 kDa)

  • Loading control (β-actin, GAPDH) to normalize expression

• For immunofluorescence:

  • DAPI nuclear counterstain to assess cellular morphology

  • F-actin staining to validate cortical co-localization

• For immunoprecipitation:

  • Input sample (pre-IP lysate)

  • Non-specific IgG IP performed in parallel

Incorporating these controls ensures experimental reliability and facilitates accurate interpretation of results across different applications.

What emerging techniques might enhance the study of APC2 function and localization?

Several cutting-edge approaches hold promise for advancing APC2 research:

• Super-resolution microscopy technologies:

  • STORM/PALM for nanoscale resolution of APC2 localization relative to cytoskeletal structures

  • Live-cell super-resolution to track dynamic changes in APC2 cortical association

• Optogenetic approaches:

  • Light-inducible APC2 recruitment to specific cellular compartments

  • Optogenetic control of domain interactions to dissect function in real-time

• Proximity labeling advancements:

  • TurboID or miniTurbo fusions for rapid biotin labeling of APC2 interaction partners

  • Domain-specific BioID to map spatial interactomes at the cortex versus cytoplasm

• CRISPR-based genomic approaches:

  • CRISPR activation/interference to modulate endogenous APC2 expression

  • Knock-in of fluorescent tags at the endogenous locus for physiological expression levels

  • Domain-specific mutations to dissect function in endogenous context

• Cryo-electron microscopy:

  • Structural determination of APC2 complexes to understand conformational changes

  • Visualization of APC2 integration into larger macromolecular assemblies

• Integrative omics approaches:

  • Correlation of APC2 interactome data with phosphoproteomics and transcriptomics

  • Computational modeling of APC2 functional networks in cytoskeletal versus Wnt contexts

These emerging technologies will enable more precise dissection of the dual roles of APC2 in actin organization and Wnt signaling regulation, potentially revealing novel therapeutic targets.

How might studying APC2 in disease models advance our understanding of its function?

Investigating APC2 in disease contexts offers valuable insights:

• Cancer research opportunities:

  • Analyze APC2 expression and localization in tumors with aberrant Wnt signaling

  • Investigate compensatory roles of APC2 in tumors with APC1 mutations

  • Examine correlation between cytoskeletal disruption and APC2 mislocalization in metastasis

• Neurodevelopmental disorder applications:

  • Study APC2 function in neuronal migration and axon guidance

  • Investigate APC2 roles in synapse formation and maintenance

  • Explore connections between APC2 and cytoskeletal abnormalities in neurodevelopmental conditions

• Embryonic development models:

  • Further explore APC2 functions in Drosophila embryogenesis

  • Extend to vertebrate models focusing on actin-dependent morphogenetic processes

  • Study tissue-specific requirements for cortical versus cytoplasmic APC2

• Therapeutic targeting potential:

  • Develop domain-specific inhibitors to selectively disrupt cytoskeletal versus Wnt functions

  • Explore methods to modulate APC2 localization as a therapeutic approach

  • Investigate synthetic lethality approaches in APC-mutant cancers

• Translational research directions:

  • Correlate APC2 antibody staining patterns with clinical outcomes in cancer

  • Develop diagnostic applications based on APC2 localization in patient samples

  • Create biomarker panels incorporating APC2 status for personalized medicine approaches