CACYBP Antibody

What is CACYBP and why is it significant in research?

CACYBP (calcyclin binding protein) is a 228 amino acid protein with a calculated molecular weight of 26 kDa, although it is typically observed at 26-30 kDa in experimental contexts . It plays a critical role in calcium-dependent ubiquitination and subsequent proteasomal degradation of target proteins, likely serving as a molecular bridge in ubiquitin E3 complexes. Its significance lies in its participation in the ubiquitin-mediated degradation of beta-catenin (CTNNB1) . CACYBP has been extensively studied in various cellular processes and has generated interest due to its expression in cancer cell lines, including gastric cancer .

What types of CACYBP antibodies are available for research, and how should I choose between them?

Researchers can choose between polyclonal and monoclonal CACYBP antibodies, each with distinct advantages:

Polyclonal antibodies (e.g., 11745-1-AP) are rabbit-derived and recognize multiple epitopes, providing high sensitivity but potentially lower specificity . They are suitable for multiple applications including WB, IHC, IF/ICC, FC, and IP.

Monoclonal antibodies (e.g., 68161-1-Ig, BD1) recognize single epitopes, offering higher specificity and consistency between batches . The monoclonal antibody BD1 (IgG1 isotype) has been specifically validated to recognize CACYBP in both native and denatured forms from human gastric cancer cell lines .

Selection should be based on your experimental requirements:

• For high sensitivity detection across multiple species (human, mouse, rat), consider polyclonal antibodies

• For consistent results in long-term studies, particularly when specificity is paramount, monoclonal antibodies are preferable

• If you're working with gastric cancer cell lines, the BD1 monoclonal antibody has been specifically validated for this application

What are the optimal dilutions for different experimental applications of CACYBP antibodies?

Optimal dilution ratios vary by application and specific antibody. Based on validated protocols, the following dilutions are recommended:

For polyclonal antibody (11745-1-AP) :

For monoclonal antibody (68161-1-Ig) :

It is strongly recommended to titrate these antibodies in each specific experimental system to determine optimal conditions, as results can be sample-dependent .

What is the proper methodology for performing antigen retrieval when using CACYBP antibodies in IHC?

For optimal antigen retrieval when using CACYBP antibodies in immunohistochemistry (IHC), the following protocol is recommended:

Primary method: Use TE buffer at pH 9.0 for antigen retrieval, as this has been validated for both polyclonal (11745-1-AP) and monoclonal (68161-1-Ig) antibodies .

Alternative method: If the primary method yields suboptimal results, citrate buffer at pH 6.0 can be used as an alternative antigen retrieval solution .

The appropriate antigen retrieval method is crucial for exposing antibody binding sites that may be masked during fixation processes. For CACYBP detection in human colon cancer tissue and mouse brain tissue, the TE buffer method has been specifically validated . Optimization of heating time, temperature, and buffer concentration may be necessary depending on tissue type and fixation conditions.

How should CACYBP antibodies be stored to maintain optimal activity?

To maintain optimal activity of CACYBP antibodies, follow these storage guidelines:

• Store antibodies at -20°C in the buffer provided (typically PBS with 0.02% sodium azide and 50% glycerol, pH 7.3) .

• Antibodies are stable for one year after shipment when stored properly at -20°C .

• Unlike some antibodies, aliquoting is unnecessary for CACYBP antibodies stored at -20°C, which simplifies laboratory protocols and reduces the risk of contamination through repeated freeze-thaw cycles .

• Before use, allow the antibody to equilibrate to room temperature and mix gently to ensure homogeneity.

• After use, promptly return the antibody to -20°C storage to prevent degradation.

Following these storage recommendations will help maintain antibody performance across experimental applications and extend the usable lifetime of these research reagents.

What are the common causes of variable band size when detecting CACYBP in Western blots?

When detecting CACYBP in Western blots, researchers may observe bands between 26-30 kDa rather than precisely at the calculated 26 kDa molecular weight . This variability can be attributed to several factors:

• Post-translational modifications: CACYBP is known to be phosphorylated on serine residues, particularly after retinoic acid induction or in high calcium environments . This phosphorylation can increase the apparent molecular weight.

• Tissue/cell type variations: The observed molecular weight of CACYBP may vary slightly between different cell lines and tissue types. The antibody has been validated to detect CACYBP in various tissues including HeLa cells, mouse and rat brain tissue, and liver tissue .

• Protein degradation: Incomplete protease inhibition during sample preparation can result in partial degradation products.

• Gel concentration and running conditions: Different percentages of polyacrylamide and varying running conditions can affect the apparent molecular weight.

To address these issues:

• Include appropriate phosphatase inhibitors if studying phosphorylated forms

• Ensure complete protease inhibition during sample preparation

• Use positive controls from validated sources (HeLa cells are recommended)

• Optimize gel concentration based on your specific experimental requirements

How can I optimize immunofluorescence staining to distinguish between nuclear and cytoplasmic CACYBP localization?

Optimizing immunofluorescence staining to distinguish between nuclear and cytoplasmic CACYBP localization requires careful attention to several methodological aspects:

• Calcium considerations: CACYBP localization is calcium-dependent. At low calcium concentrations, it is primarily cytoplasmic, while after retinoic acid induction and calcium increase, it localizes to both nucleus and cytoplasm . Consider controlling or measuring calcium levels in your experimental system.

• Fixation method optimization:

  • For preserving cytoplasmic CACYBP: 4% paraformaldehyde fixation (10-15 minutes at room temperature)

  • For preserving nuclear CACYBP: Methanol fixation (-20°C for 10 minutes) often better preserves nuclear proteins

• Permeabilization considerations: Use 0.1-0.2% Triton X-100 for sufficient nuclear permeabilization without excessive cytoplasmic extraction.

• Antibody selection and dilution: For IF/ICC applications, use polyclonal antibodies at 1:50-1:500 dilution or monoclonal antibodies at 1:400-1:1600 dilution . The lower end of the dilution range (more concentrated antibody) may be necessary for detecting nuclear CACYBP.

• Co-staining strategy: Use DAPI for nuclear counterstaining and phalloidin for cytoskeletal counterstaining to accurately distinguish cellular compartments.

• Confocal microscopy: For definitive localization, confocal microscopy with Z-stack acquisition is recommended to distinguish between true nuclear localization and cytoplasmic protein overlying the nucleus.

HeLa and HEK-293 cells have been validated for successful CACYBP immunofluorescence detection and can serve as positive controls .

How can CACYBP antibodies be utilized to study protein-protein interactions in ubiquitin-proteasome pathways?

CACYBP antibodies can be leveraged to study protein-protein interactions in ubiquitin-proteasome pathways through several advanced methodological approaches:

• Co-immunoprecipitation (Co-IP): CACYBP antibodies have been validated for immunoprecipitation in mouse brain tissue and HeLa cells , making them suitable for pulling down CACYBP and its interacting partners. This approach can help identify:

  • Interactions with S100 family proteins (S100A1, S100A6, S100B, S100P, and S100A12)

  • Components of ubiquitin E3 complexes where CACYBP serves as a molecular bridge

  • Beta-catenin (CTNNB1) interactions during ubiquitin-mediated degradation pathways

• Proximity Ligation Assay (PLA): Combine CACYBP antibodies with antibodies against suspected interaction partners to visualize and quantify specific protein-protein interactions in situ with spatial resolution.

• FRET/BRET analysis: Use CACYBP antibodies to validate interaction partners identified through fluorescence or bioluminescence resonance energy transfer experiments.

• Calcium-dependent interaction studies: Since CACYBP interactions are calcium-dependent , design experiments with varying calcium concentrations to study how calcium levels affect CACYBP's role in ubiquitin-proteasome pathways.

• Phosphorylation-specific studies: Combine CACYBP antibodies with phospho-specific antibodies to investigate how phosphorylation affects CACYBP's interactions within ubiquitin-proteasome complexes.

For immunoprecipitation experiments, use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate to achieve optimal results .

What approaches can be used to study CACYBP phosphorylation states and their functional significance?

Studying CACYBP phosphorylation states and their functional significance requires sophisticated methodological approaches:

• Phosphorylation-state specific antibodies: While the current literature doesn't mention phospho-specific CACYBP antibodies, custom phospho-antibodies could be developed targeting known serine phosphorylation sites.

• Phosphatase treatment experiments: Compare Western blot migration patterns of CACYBP before and after treatment with lambda phosphatase to identify mobility shifts caused by phosphorylation.

• Mass spectrometry approach:

  • Immunoprecipitate CACYBP using validated antibodies from cells under different conditions (e.g., with/without retinoic acid induction, varying calcium concentrations)

  • Perform tryptic digestion and phosphopeptide enrichment (using TiO₂ or IMAC)

  • Analyze by LC-MS/MS to identify and quantify phosphorylation sites

• Mutational analysis: Generate serine-to-alanine (phospho-deficient) or serine-to-glutamate (phospho-mimetic) CACYBP mutants and analyze their:

  • Subcellular localization (nuclear vs. cytoplasmic distribution)

  • Interaction with S100 family proteins

  • Function in ubiquitin-mediated degradation of beta-catenin

• Correlative microscopy: Combine immunofluorescence of CACYBP (using validated dilutions of 1:50-1:500 for polyclonal or 1:400-1:1600 for monoclonal antibodies) with phospho-specific staining to correlate phosphorylation state with subcellular localization.

Remember that CACYBP phosphorylation is induced by retinoic acid or high calcium concentrations , so experimental designs should include these conditions as positive controls.

How should researchers interpret CACYBP expression patterns across different cancer cell lines?

When interpreting CACYBP expression patterns across different cancer cell lines, researchers should consider several methodological and contextual factors:

• Validated detection in multiple cancer cell lines: CACYBP antibodies have been validated for detection in diverse cancer cell lines including HeLa, HCT 116, 4T1, LNCaP, HEK-293, Jurkat, and K-562 cells . This provides a foundation for comparative studies.

• Quantitative analysis approach:

  • For relative quantification: Western blot analysis using standardized protocols with dilutions of 1:1000-1:6000 (polyclonal) or 1:5000-1:50000 (monoclonal)

  • For absolute quantification: Consider ELISA-based approaches

  • Normalize expression to appropriate housekeeping proteins that remain constant across the cancer cell lines being compared

• Subcellular localization considerations: CACYBP can localize to both cytoplasm and nucleus depending on calcium levels and cellular state . When comparing cell lines:

  • Perform subcellular fractionation to distinguish between cytoplasmic and nuclear pools

  • Use immunofluorescence (at validated dilutions) to visualize distribution patterns

• Correlative analysis with cancer phenotypes:

  • Integrate CACYBP expression data with proliferation rates, invasion capacity, and other cancer hallmarks

  • Consider the relationship between CACYBP expression and beta-catenin levels, given CACYBP's role in beta-catenin degradation

• Calcium signaling context: Since CACYBP function is calcium-dependent , consider the calcium signaling characteristics of different cancer types when interpreting expression patterns.

The monoclonal antibody BD1 has been specifically validated for detecting CACYBP in gastric cancer cell lines in both native and denatured forms , making it particularly valuable for gastric cancer research.

What is the current understanding of CACYBP's role in neurodegenerative diseases and how can researchers investigate this further?

The current understanding of CACYBP's role in neurodegenerative diseases is still emerging, but researchers can investigate this relationship through several methodological approaches:

• Brain tissue expression analysis: CACYBP antibodies have been validated for detection in mouse and rat brain tissues , providing tools for investigating expression patterns in:

  • Different brain regions

  • Various neurodegenerative disease models

  • Human postmortem brain samples from patients with neurodegenerative conditions

• Neuron-specific investigations:

  • Primary neuron cultures: Use immunofluorescence (at dilutions of 1:50-1:500 for polyclonal or 1:400-1:1600 for monoclonal antibodies) to examine CACYBP expression and localization in neurons

  • Brain organoids: Apply similar immunostaining techniques to 3D brain organoid models of neurodegenerative diseases

• Calcium signaling connection: Given that CACYBP function is calcium-dependent and calcium dysregulation is implicated in many neurodegenerative diseases:

  • Design experiments that modulate calcium levels while monitoring CACYBP localization and function

  • Investigate CACYBP's relationship with calcium-binding S100 proteins in neuronal contexts

• Proteasomal degradation pathway: Since CACYBP participates in ubiquitin-mediated protein degradation , and proteasome dysfunction is common in neurodegenerative diseases:

  • Examine CACYBP's interaction with disease-relevant proteins that undergo ubiquitin-mediated degradation

  • Investigate whether CACYBP function is altered in conditions of proteasomal stress

• Phosphorylation dynamics: CACYBP can be phosphorylated on serine residues , which may affect its function in neuronal contexts:

  • Compare phosphorylation states in healthy versus diseased brain tissues

  • Investigate kinase pathways that might regulate CACYBP phosphorylation in neurons

For immunohistochemistry of brain tissue samples, researchers should use the validated antigen retrieval method with TE buffer at pH 9.0 and antibody dilutions of 1:1000-1:4000 (polyclonal) or 1:500-1:2000 (monoclonal) .

How can CACYBP antibodies be used in live-cell imaging experiments to track dynamic processes?

While traditional applications of CACYBP antibodies focus on fixed cells, adapting them for live-cell imaging requires specialized approaches:

• Antibody fragment preparation:

  • Generate Fab fragments from whole CACYBP antibodies to improve cellular penetration

  • Consider single-domain antibodies (nanobodies) as alternatives for live-cell applications

• Fluorescent labeling strategies:

  • Directly conjugate purified CACYBP antibodies or fragments with bright, photostable fluorophores (e.g., Alexa Fluor dyes)

  • Use antibody labeling kits that minimize the impact on antibody functionality

  • Calculate optimal fluorophore-to-protein ratios to avoid quenching effects

• Cell delivery methods:

  • Microinjection: Precise delivery but low throughput

  • Cell-penetrating peptides: Conjugate to antibody fragments for enhanced uptake

  • Electroporation: Higher throughput but potential cell stress

  • Bead-loading: Mechanical delivery for adherent cells

• Experimental design considerations:

  • Pre-validate antibody specificity using fixed-cell immunofluorescence at recommended dilutions (1:50-1:500 for polyclonal or 1:400-1:1600 for monoclonal)

  • Include appropriate controls (non-binding antibody fragments with the same fluorophore)

  • Minimize laser power and exposure times to reduce phototoxicity

  • Consider using spinning disk confocal microscopy for reduced phototoxicity

• Alternative genetic approaches:

  • As a complementary approach, consider CRISPR-mediated endogenous tagging of CACYBP with fluorescent proteins

  • Validate such constructs using the well-characterized CACYBP antibodies to ensure proper localization and function

Due to CACYBP's calcium-dependent localization , live-cell experiments should include calcium monitoring to correlate CACYBP dynamics with calcium fluctuations.

What are the best practices for using CACYBP antibodies in multi-color flow cytometry experiments?

For optimal results when incorporating CACYBP antibodies into multi-color flow cytometry panels:

• Antibody selection and preparation:

  • For intracellular CACYBP detection, use the validated flow cytometry antibody at 0.25 μg per 10^6 cells in 100 μl suspension

  • Consider using directly conjugated antibodies where available to minimize background and reduce protocol complexity

• Panel design considerations:

  • Place CACYBP detection in an appropriate fluorochrome channel based on its expected expression level (brighter fluorochromes for lower expression)

  • Account for CACYBP's intracellular location when designing the staining sequence

  • Include appropriate isotype controls matched to antibody class (IgG2a for monoclonal, IgG for polyclonal)

• Optimized staining protocol:

  • Cell fixation: Use 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilization: Select reagents based on target localization (Triton X-100 or saponin for cytoplasmic CACYBP, methanol for nuclear CACYBP)

  • Blocking: Use 1-5% BSA or 5-10% serum from the same species as the secondary antibody

  • Staining sequence: Surface markers first, followed by fixation, permeabilization, and intracellular CACYBP staining

• Controls and validation:

  • Include single-color controls for compensation

  • Use FMO (Fluorescence Minus One) controls to set accurate gates

  • Validate with positive control samples (HeLa cells have been confirmed positive for CACYBP)

  • Consider parallel Western blot analysis to confirm specificity at the expected 26-30 kDa molecular weight

• Analysis considerations:

  • Gate on single, viable cells before analyzing CACYBP expression

  • When studying calcium-dependent changes in CACYBP, consider co-staining with calcium indicators

  • For phosphorylation studies, include appropriate phospho-specific markers