CCKAR Antibody

What is CCKAR and why is it important in scientific research?

CCKAR (Cholecystokinin A Receptor) is a G protein-coupled receptor that mediates pancreatic growth and enzyme secretion, as well as smooth muscle contraction of the gallbladder and stomach. It has a 1000-fold higher affinity for cholecystokinin (CCK) compared to gastrin, and modulates feeding and dopamine-induced behavior in both central and peripheral nervous systems . CCKAR has gained significant research interest due to its involvement in various pathological conditions, including being identified as a prognostic biomarker in non-small cell lung cancer (NSCLC) and its correlation with asynchronous brain metastasis . The receptor mediates its action through G proteins that activate a phosphatidylinositol-calcium second messenger system, making it a valuable target for studying cellular signaling pathways .

What are the common synonyms and alternative names for CCKAR in scientific literature?

When conducting literature searches about CCKAR, researchers should be aware of multiple synonyms used across different publications:

These alternative nomenclatures are important to consider when performing comprehensive literature reviews or database searches to ensure all relevant research is captured .

Which species do most commercial CCKAR antibodies show reactivity with?

Most commercial CCKAR antibodies demonstrate reactivity with human, mouse, rat, and chicken samples, as verified through various applications . Predicted reactivity, based on sequence homology but requiring experimental validation, extends to other species including dog, cow, sheep, pig, and horse . When selecting an antibody for research with less commonly used model organisms, it's advisable to review the antibody data sheet for cross-reactivity information or conduct preliminary validation experiments to confirm species-specificity .

What are the optimal protocols for using CCKAR antibodies in immunohistochemistry?

For optimal immunohistochemical detection of CCKAR, researchers should follow these methodological considerations:

• Antigen Retrieval: For paraffin-embedded sections, use either citrate buffer (pH 6.0) or TE buffer (pH 9.0) for antigen retrieval. Studies have shown that both buffers can be effective, though optimal conditions may be sample-dependent .

• Blocking and Primary Antibody Incubation:

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Prevent non-specific binding using 5% bovine serum albumin (BSA)

  • Incubate with primary CCKAR antibody at appropriate dilutions (commonly 1:20-1:200 for IHC-P) overnight at 4°C .

• Secondary Antibody and Detection:

  • Apply appropriate secondary antibody conjugated with streptavidin-biotin-peroxidase reagent for 30 minutes at room temperature

  • Visualize using 3,3'-diaminobenzidine (DAB) solution .

• Semi-quantification Method: For expression analysis, implement a scoring system considering both staining intensity (0-3) and positive stained cell percentage (1-4), with final scores calculated as the product of these parameters (range: 0-12) .

How should CCKAR antibody concentration be optimized for Western blotting?

For Western blotting applications with CCKAR antibodies, optimization requires systematic titration:

• Initial Dilution Range: Start with a broad dilution series (e.g., 1:300, 1:1000, 1:3000) based on manufacturer recommendations (typically 1:300-1:5000) .

• Blocking Optimization: Test different blocking agents (5% non-fat milk vs. 5% BSA) as CCKAR detection may be sensitive to blocking conditions.

• Incubation Parameters: Optimize both primary antibody incubation temperature (4°C vs. room temperature) and duration (overnight vs. 1-3 hours).

• Expected Molecular Weight: Visualize CCKAR at its calculated molecular weight of approximately 48 kDa, though post-translational modifications may affect migration patterns .

• Internal Controls: Always include positive control tissues (e.g., human stomach tissue) where CCKAR expression is well-established .

What approaches can be used for quantifying CCKAR expression in tissue samples?

Multiple quantification methods have been validated for measuring CCKAR expression levels:

• qRT-PCR Approach:

  • Extract total RNA using TRIzol reagent

  • Perform reverse transcription with appropriate kits (e.g., ReverTra kit)

  • Use SYBR-based qPCR with specific primers:

    • CCKAR forward: 5'-ATGGATGTGGTTGACAGCCTT-3'

    • CCKAR reverse: 5'-AAGCGTCTCATTTTCGAGCCC-3'

    • GAPDH (internal control) forward: 5'-GAGTCAACGGATTTGGTCGT-3'

    • GAPDH (internal control) reverse: 5'-GACAAGCTTCCCGTTCTCAG-3'

  • Analyze using the 2^-ΔΔCt method with appropriate housekeeping gene normalization .

• ELISA-Based Quantification:

  • Coat plates with diluted antigen

  • Block with 1% BSA

  • Incubate with anti-human CCKAR antibody (typically 1:1000 dilution)

  • Apply secondary antibody (1:2000)

  • Develop with substrate solution and measure absorbance at 450nm .

• Immunohistochemical Semi-Quantification:

  • Score staining intensity: 0 (negative), 1 (weak), 2 (moderate), 3 (strong)

  • Assess positive cell percentage: 1 (<25%), 2 (25-50%), 3 (50-75%), 4 (75-100%)

  • Calculate final score as product of both parameters (range: 0-12)

  • Determine expression cutoffs using receiver operating characteristic (ROC) curves .

How is CCKAR expression analyzed in cancer research and what are the methodological considerations?

CCKAR has emerged as an important biomarker in cancer research, particularly in non-small cell lung cancer (NSCLC) and gallbladder cancer (GBC):

• Expression Profiling in Tumor vs. Normal Tissue:

  • In NSCLC, CCKAR is significantly overexpressed compared to para-tumor tissues, as demonstrated by both qRT-PCR and IHC analyses

  • CCKAR primarily localizes to cytoplasm and membrane in cancer cells

  • CCK-8 (ligand) expression is typically low or undetectable in NSCLC samples .

• Correlation with Metastatic Potential:

  • CCKAR expression positively associates with asynchronous brain metastasis (BM) in NSCLC

  • Higher expression levels correlate with increased risk of developing BM

  • Statistical analyses with appropriate cutoff values help stratify patient risk groups .

• Methodological Approaches:

  • Paired analysis of primary tumors and metastatic sites

  • Comparison of expression levels between cancer and corresponding adjacent normal tissues

  • Correlation of expression with clinicopathological parameters and survival outcomes

  • Validation through multiple detection methods (IHC, qRT-PCR, ELISA) .

What controls should be included when validating a new CCKAR antibody for research?

Rigorous validation of CCKAR antibodies requires comprehensive control experiments:

• Positive Tissue Controls:

  • Human stomach tissue (established high CCKAR expression)

  • Pancreatic tissue (physiologically relevant CCKAR-expressing tissue)

  • Cell lines with known CCKAR expression (e.g., certain pancreatic or gastrointestinal cell lines) .

• Negative Controls:

  • Isotype-matched irrelevant antibodies at equivalent concentrations

  • Antibody pre-absorption with immunizing peptide

  • Tissues known to lack CCKAR expression or CCKAR-knockout tissues/cells when available.

• Technical Validation Controls:

  • Gradient dilution series to confirm specificity and optimal concentration

  • Parallel validation with multiple antibodies targeting different epitopes of CCKAR

  • Correlation of protein detection with mRNA expression data .

• Functional Validation:

  • Calcium mobilization assays (as demonstrated in chicken peripheral blood mononuclear cells)

  • Receptor signaling studies following antibody binding

  • Downstream pathway activation assessment .

How can researchers distinguish between CCKAR and related receptors in experimental systems?

Accurate discrimination between CCKAR and related receptors (particularly CCKBR/gastrin receptor) requires careful experimental design:

• Antibody Epitope Selection:

  • Choose antibodies targeting regions with minimal sequence homology between receptor subtypes

  • Verify epitope specificity through sequence alignment analysis prior to experimental use

  • Consider antibodies raised against specific amino acid regions (e.g., AA 161-200, AA 325-356) that are unique to CCKAR .

• Pharmacological Discrimination:

  • Leverage CCKAR's 1000-fold higher affinity for CCK compared to gastrin in binding studies

  • Employ selective CCKAR antagonists (e.g., devazepide) versus CCKBR antagonists in functional studies

  • Conduct competitive binding assays with labeled ligands of varying specificity.

• Molecular Validation:

  • Perform parallel detection with receptor subtype-specific primers in PCR

  • Use siRNA knockdown specific to CCKAR to confirm antibody specificity

  • Consider dual labeling approaches in microscopy to evaluate co-localization patterns.

What are common issues encountered with CCKAR antibodies in IHC and how can they be resolved?

Researchers frequently encounter several challenges when using CCKAR antibodies for immunohistochemistry:

• High Background Staining:

  • Problem: Non-specific binding resulting in excessive background

  • Solution: Optimize blocking (increase BSA concentration to 5%), extend blocking time (60+ minutes), and introduce additional washing steps with 0.1% Tween-20 in PBS .

• Weak or Absent Signal:

  • Problem: Insufficient antigen retrieval or suboptimal antibody concentration

  • Solution: Compare different antigen retrieval methods (citrate buffer pH 6.0 vs. TE buffer pH 9.0), extend retrieval time, and test concentration range between 1:20-1:200 for optimal signal-to-noise ratio .

• Inconsistent Staining Patterns:

  • Problem: Variability in tissue fixation or processing

  • Solution: Standardize fixation protocols (duration and fixative composition), ensure consistent sectioning thickness (4-5μm recommended), and maintain precise incubation times and temperatures .

• Non-specific Nuclear Staining:

  • Problem: Unexpected nuclear localization when CCKAR is primarily membrane/cytoplasmic

  • Solution: Verify antibody specificity, optimize permeabilization conditions, and consider using membrane fraction enrichment protocols for validation studies.

How can researchers validate CCKAR antibody specificity for flow cytometry applications?

For flow cytometry applications, CCKAR antibody validation requires specific considerations:

• Blocking Strategy:

  • Pre-incubate cells with 1% FBS in PBS containing 0.5% FBS and 0.01% NaN₃ to minimize non-specific binding

  • Consider Fc receptor blocking when working with immune cells .

• Titration Approach:

  • Test antibody across concentration range (typically 1:20-1:100 dilution)

  • Determine optimal concentration by evaluating separation between positive and negative populations

  • Confirm specificity using appropriate isotype controls matched for concentration .

• Specificity Controls:

  • Include CCKAR-negative cell populations as internal negative controls

  • Perform pre-absorption controls with immunizing peptide

  • Compare staining patterns with alternative CCKAR antibodies targeting different epitopes

  • Use cell lines with confirmed CCKAR knockdown/knockout when available .

• Multi-parameter Validation:

  • Combine CCKAR staining with cell-type specific markers (e.g., KUL01 for monocytes)

  • Confirm CCKAR expression changes following relevant physiological stimuli

  • Correlate flow cytometry results with other detection methods (Western blot, qRT-PCR) .

What is the significance of different CCKAR antibody immunogens and how do they impact experimental outcomes?

The immunogen used to generate CCKAR antibodies significantly influences their performance characteristics:

• Peptide vs. Protein Immunogens:

  • Synthetic Peptide Immunogens: Most commercial CCKAR antibodies utilize KLH-conjugated synthetic peptides derived from specific CCKAR regions (e.g., AA 161-200, AA 325-356)

  • Advantages: High specificity for the target epitope, reduced cross-reactivity

  • Limitations: May recognize denatured but not native protein forms

  • Applications: Optimal for Western blotting and IHC of fixed tissues, may be suboptimal for applications requiring recognition of native conformations .

• Epitope Location Considerations:

  • N-terminal: Antibodies targeting N-terminal regions may detect post-translationally modified or truncated forms

  • Internal Regions: Antibodies recognizing internal domains (e.g., AA 161-200) are commonly used across multiple applications

  • C-terminal: C-terminal targeting antibodies may be affected by protein-protein interactions or post-translational modifications .

• Cross-reactivity Assessment:

  • Sequence alignment analysis between target epitope and homologous proteins is essential

  • Immunogen selection influences cross-species reactivity profiles

  • Domain-specific antibodies may exhibit differential reactivity in various experimental contexts .

• Application-Specific Considerations:

  • For membrane protein detection in flow cytometry or live-cell imaging, antibodies recognizing extracellular domains are preferred

  • For signaling studies, antibodies against intracellular regulatory domains may provide functional insights

  • For protein-protein interaction studies, consider epitope accessibility in native protein complexes .