CAP2 Antibody

What criteria should researchers use when selecting a CAP2 antibody for their experiments?

When selecting a CAP2 antibody, researchers should consider:

• Antibody type: Monoclonal antibodies like CAP2 Antibody (A-5) offer high specificity, while polyclonal antibodies may provide stronger signals through multiple epitope recognition .

• Species reactivity: Verify cross-reactivity with your experimental model. Some antibodies detect CAP2 in human, mouse, and rat samples, while others have limited species reactivity .

• Application compatibility: Confirm the antibody is validated for your intended application (WB, IHC, IF, IP, ELISA). For example, CAP2 antibody (15865-1-AP) works in WB, IHC, IF/ICC, IP, CoIP, and ELISA applications .

• Target region: Consider antibodies targeting different regions of CAP2 based on your research question. For instance, ab236590 targets amino acids 200 to C-terminus of human adenylyl cyclase-associated protein 2 .

What validation methods should be employed to confirm CAP2 antibody specificity?

Robust validation of CAP2 antibodies should include:

• Knockout/knockdown verification: Test antibody on CAP2 knockout/knockdown samples to confirm specificity. Multiple studies have validated CAP2 antibodies using this approach .

• Multiple detection methods: Confirm CAP2 detection using orthogonal techniques (e.g., WB, IF, IHC) to strengthen confidence in antibody specificity .

• Co-localization studies: Verify CAP2 localization patterns match known distribution patterns in tissues. CAP2 is predominantly expressed in skin, brain, heart, skeletal muscle, and shows specific cellular localization patterns .

• Molecular weight verification: Confirm detection at the expected molecular weight (~53 kDa for CAP2) .

What are the optimal sample preparation protocols for CAP2 detection in different experimental systems?

Sample preparation varies by application:

For Western Blotting:

• Denature protein extracts at 95°C in Laemmli buffer

• Separate by SDS-PAGE and blot onto PVDF membrane

• Block for 1 hour followed by overnight primary antibody incubation at 4°C

• Recommended dilutions: 1:1000-1:6000 for polyclonal (15865-1-AP) and 1:2000-1:10000 for monoclonal (67412-1-Ig) antibodies

For Immunofluorescence:

• Fix cells using 4% paraformaldehyde

• For CAP2 detection in neuronal samples, co-staining with markers like VGLUT1 (for excitatory presynaptic terminals) or PSD-95 helps confirm localization

• Recommended dilution: 1:50-1:500 for polyclonal antibody (15865-1-AP)

For Immunohistochemistry:

• For paraffin-embedded tissues, antigen retrieval is crucial; use TE buffer pH 9.0 or citrate buffer pH 6.0

• For CAP2 detection in brain tissue, embryonic (E18) and postnatal (P30, P365) stages can show different expression patterns

• Recommended dilution: 1:20-1:200 for polyclonal antibody (15865-1-AP)

How can researchers quantify CAP2 protein levels in relation to actin dynamics?

To study CAP2's role in actin dynamics:

• G-actin/F-actin fractionation assay:

  • Lyse cells in a buffer that stabilizes both G-actin and F-actin forms

  • Separate by ultracentrifugation to fractionate G-actin (supernatant) from F-actin (pellet)

  • Analyze fractions by western blotting using CAP2 and actin antibodies

  • This approach can quantify the G-actin/F-actin ratio in CAP2 overexpression or knockout systems

• Actin dynamics measurement:

  • Use fluorescence recovery after photobleaching (FRAP) to measure actin incorporation at pointed ends

  • CAP2 overexpression results in lower percentage of F-actin (49.5 ± 3.4%) compared to controls (60.6 ± 3.7%)

  • FRAP analysis can demonstrate that CAP2 specifically inhibits actin incorporation at pointed ends but not barbed ends

• Cofilin activity correlation:

  • Measure phospho-cofilin/total cofilin ratios as CAP2 regulates cofilin activity

  • Analyze LIMK2, phospho-LIMK1/2, TESK1, ROCK1, and chronophin levels to understand the mechanistic pathway

How should researchers design experiments to study CAP2's role in hepatocellular carcinoma (HCC)?

Based on established research, optimal approaches include:

What is the methodological approach for investigating CAP2's role in cardiac conduction and sudden cardiac death?

To study CAP2's cardiac functions:

• Generate appropriate genetic models:

  • Use conditional knockout systems (e.g., loxP-CAP2 mice crossed with Myh6Cre mice for cardiomyocyte-specific deletion)

  • Validate knockout efficiency through qPCR and western blot analysis

• Cardiac phenotype characterization:

  • Perform echocardiography to assess cardiac function

  • Monitor for sudden cardiac death events in CAP2-deficient models

  • Conduct ECG studies to identify conduction defects

• Molecular mechanism investigation:

  • Analyze actin dynamics in cardiomyocytes using G-actin/F-actin fractionation

  • Investigate CAP2 interaction with cardiac-specific proteins

  • Study calcium handling in CAP2-deficient cardiomyocytes

How can researchers optimize CAP2 co-immunoprecipitation experiments to study protein-protein interactions?

For successful CAP2 co-immunoprecipitation:

• Antibody selection:

  • Choose antibodies validated for IP applications (e.g., CAP2 Antibody A-5 or 15865-1-AP)

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

• Lysis buffer optimization:

  • Use buffers containing mild detergents (e.g., 1% NP-40 or 0.5% Triton X-100)

  • Include protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if studying phosphorylation-dependent interactions

• CAP2 domain-specific interactions:

  • Design experiments to study specific domains:

    • N-terminal region (1-164) mediates CAP2 self-association

    • Deletion of amino acids 1-164 significantly reduces self-association

    • C-CAP/cofactor C-like domain is essential for interaction with actin

• Controls:

  • Include isotype control antibodies

  • Use CAP2 knockout/knockdown samples as negative controls

  • Include input samples to confirm protein expression

What methodological considerations should be taken when studying CAP2's role in neuronal actin dynamics and dendritic spine morphology?

For neuronal CAP2 studies:

• Neuronal culture system selection:

  • Primary cortical neurons show CAP2 localization in dendritic shafts and mature spines

  • Co-label with VGLUT1 to identify excitatory presynaptic terminals

  • Co-label with PSD-95 to localize CAP2 at postsynaptic densities

• Dendritic spine analysis:

  • Use high-resolution imaging techniques (confocal, super-resolution)

  • Quantify spine density, morphology changes (mushroom, thin, stubby types)

  • In CAP2-deficient mice, dendritic complexity and spine morphology are altered

• Actin dynamics visualization:

  • Use fluorescent actin probes (Lifeact, phalloidin) to co-visualize with CAP2

  • Perform FRAP experiments to measure actin turnover rates in dendritic spines

  • Analyze cofilin activity as CAP2 regulates cofilin localization in spines

• Synaptic function correlation:

  • Measure synaptic protein levels (GluA1, PSD-95)

  • Correlate CAP2 expression with electrophysiological measurements

  • Analyze behavioral outcomes in CAP2-deficient animal models

How should researchers address inconsistent CAP2 antibody staining patterns across different tissues?

When encountering variable staining:

• Validate tissue-specific expression patterns:

  • CAP2 shows differential expression across tissues and developmental stages

  • Expression levels are relatively low in olfactory bulb and hippocampus at E18 but increase at P30 and P365

  • Predominantly expressed in skin, brain, heart, skeletal muscle

• Optimize tissue-specific protocols:

  • For brain tissue: Consider developmental stage-specific fixation protocols

  • For muscle tissue: Extended fixation may be required for proper antibody penetration

  • For cancer tissues: Compare with matched normal tissues to establish baseline expression

• Epitope accessibility considerations:

  • Different fixation methods can affect CAP2 epitope exposure

  • For IHC, test both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

  • For difficult tissues, consider alternative antibodies targeting different CAP2 epitopes

• Cross-validation approaches:

  • Use both monoclonal and polyclonal antibodies to confirm staining patterns

  • Validate with RNA expression data (e.g., in situ hybridization)

  • Confirm with western blot analysis of the same tissues

What are the critical factors to consider when interpreting CAP2 expression data in the context of actin regulatory mechanisms?

For accurate interpretation:

• Consider CAP2 vs. CAP1 expression:

  • CAP1 and CAP2 show different tissue distribution patterns

  • CAP1 is more uniformly expressed across brain regions at P30 and P365, while CAP2 shows regional specificity

  • CAP2 mRNA expression positively correlates with CAP1 mRNA expression in some contexts

• Evaluate related actin regulatory proteins:

  • Measure cofilin, phospho-cofilin, LIMK2, and TESK1 levels alongside CAP2

  • CAP2 deletion alters G-actin/F-actin ratio, shifting equilibrium towards G-actin

  • CAP2 overexpression results in decreased F-actin percentage (49.5 ± 3.4%) compared to controls (60.6 ± 3.7%)

• Context-dependent functions:

  • In cardiomyocytes: CAP2 regulates actin pointed end dynamics and myofibrillogenesis

  • In neurons: CAP2 affects dendritic spine morphology and cofilin localization

  • In hepatocellular carcinoma: CAP2 overexpression correlates with poor prognosis

• Quantitative analysis approaches:

  • Use G-actin/F-actin fractionation assays to quantify the effect of CAP2 on actin dynamics

  • Apply FRAP analysis to measure actin incorporation rates at pointed and barbed ends

  • Consider subcellular localization in interpretation (cytoplasmic, nuclear, or membrane-associated)