CAMTA1 Antibody

What is CAMTA1 and what is its biological significance?

CAMTA1 is a calmodulin-binding transcription activator with a calculated molecular weight of approximately 183,672 Da . The protein functions as a potential tumor suppressor candidate located on chromosome 1p36 . In normal tissues, CAMTA1 expression is primarily limited to the brain, distinguishing it from its fusion partner WWTR1, which is expressed in many different cell types .

CAMTA1 has demonstrated growth-suppressive properties in experimental models. When induced in neuroblastoma SH-EP cells, CAMTA1 significantly decreases colony formation ability and growth rate, suggesting its role in regulating cell proliferation . Research has shown that CAMTA1 induction increases the proportion of cells in G1/G0 phase (from 59.3% to 69.9%) with a concomitant decrease in S-phase cells (from 29.2% to 19.5%), indicating its involvement in cell cycle regulation .

What types of CAMTA1 antibodies are available for research?

Several types of CAMTA1 antibodies are available for research purposes, with variations in their target epitopes, host species, and applications:

Additionally, various conjugated versions of CAMTA1 antibodies are available, including those labeled with HRP, FITC, biotin, and various Alexa Fluor dyes, enabling diverse detection methods .

What are the recommended applications for CAMTA1 antibodies?

CAMTA1 antibodies are validated for multiple research applications, with specific recommendations for each technique:

• Western Blotting (WB): Several CAMTA1 antibodies are validated for western blot applications, with recommended dilutions ranging from 1:500 to 1:2000 . This technique is valuable for detecting the fusion protein in tumor samples and confirming specificity.

• Enzyme-Linked Immunosorbent Assay (ELISA): CAMTA1 antibodies can be used in ELISA with recommended dilutions of 1:5000 to 1:20000, providing a quantitative method for measuring CAMTA1 protein levels .

• Immunohistochemistry (IHC): Polyclonal antibodies directed against the C-terminus of CAMTA1 have proven useful for diagnostic purposes in tissue sections, particularly for identifying EHE through nuclear CAMTA1 expression .

• Immunofluorescence (IF): Some CAMTA1 antibodies are specifically validated for immunofluorescence applications, allowing for subcellular localization studies and co-localization with other proteins of interest .

How should CAMTA1 antibodies be stored and handled?

Proper storage and handling of CAMTA1 antibodies are crucial for maintaining their specificity and reactivity:

For long-term storage, CAMTA1 antibodies should be kept at -20°C, where they can remain stable for up to one year . For frequent use and short-term storage (up to one month), antibodies can be stored at 4°C to avoid repeated freeze-thaw cycles .

Researchers should be aware that most CAMTA1 antibodies are supplied in liquid form in PBS containing preservatives such as 50% glycerol and 0.02% sodium azide . These additives help maintain antibody stability but may interfere with certain applications. When planning experiments, consider that sodium azide inhibits horseradish peroxidase activity and should be removed for certain detection methods.

How can CAMTA1 antibodies be used for studying epithelioid hemangioendothelioma (EHE)?

CAMTA1 antibodies have proven highly valuable for studying and diagnosing epithelioid hemangioendothelioma (EHE), a malignant endothelial neoplasm characterized by recurrent WWTR1-CAMTA1 gene fusions. Research has validated that polyclonal antibodies directed against the C-terminus of CAMTA1 can effectively identify EHE cases through immunohistochemistry .

In a comprehensive study of 204 tumors, 86% (51/59) of EHE cases demonstrated diffuse nuclear staining for CAMTA1, including 92% (44/48) of cases with conventional histology and 64% (7/11) of cases with "malignant" histology . Importantly, with the exception of one case previously diagnosed as epithelioid angiosarcoma, all other epithelioid mesenchymal neoplasms examined were negative for CAMTA1 .

The methodological approach involves:

• Whole-tissue section immunohistochemistry using anti-CAMTA1 polyclonal antibody

• Evaluation of nuclear staining pattern and intensity

• Correlation with histological features and clinical information

• Confirmatory testing for TFE3 in CAMTA1-negative cases (6 of 8 CAMTA1-negative EHE cases were positive for TFE3)

What methodological considerations are important for CAMTA1 immunohistochemistry?

When performing CAMTA1 immunohistochemistry for diagnostic or research purposes, several methodological considerations are crucial:

• Antibody Selection: The choice of anti-CAMTA1 antibody is critical, as different polyclonal antibodies directed against the C-terminus have shown varying results. Some earlier antibodies reported widespread expression in normal tissues and diverse tumor types, while newer antibodies demonstrate greater specificity for EHE .

• Control Tissues: Given that normal CAMTA1 expression is limited to the brain, appropriate positive controls (brain tissue or known EHE cases) and negative controls (non-EHE epithelioid tumors) should be included in every staining run.

• Staining Pattern Interpretation: Nuclear staining is the hallmark of CAMTA1 positivity in EHE. Researchers should focus on diffuse nuclear staining patterns rather than cytoplasmic or membranous staining, which may represent non-specific binding .

• Complementary Testing: For cases with suspected EHE but negative CAMTA1 staining, testing for TFE3 is recommended, as a small subset of EHE cases (<5%) harbor YAP1-TFE3 fusion genes instead of WWTR1-CAMTA1 .

How can researchers optimize Western blotting protocols for CAMTA1 detection?

Optimizing Western blotting protocols for CAMTA1 detection requires careful consideration of several factors:

• Sample Preparation: Given CAMTA1's high molecular weight (approximately 183,672 Da), use low percentage gels (6-8%) or gradient gels to ensure adequate separation . Complete protein denaturation is essential; use strong lysis buffers containing SDS and DTT or β-mercaptoethanol.

• Transfer Conditions: For high molecular weight proteins like CAMTA1, extended transfer times or semi-dry transfer systems may be necessary. Consider using PVDF membranes rather than nitrocellulose for better protein retention.

• Antibody Dilution: Start with the manufacturer's recommended dilution range (1:500-1:2000 for WB) and optimize based on signal-to-noise ratio . Always perform a dilution series during protocol optimization.

• Blocking and Washing: Use 5% non-fat dry milk or BSA in TBST for blocking. Include multiple thorough washing steps with TBST to reduce background signal, which is particularly important when working with polyclonal antibodies.

• Detection System: Enhanced chemiluminescence (ECL) systems are typically suitable, but for low expression levels, consider more sensitive detection methods such as ECL Plus or femto-sensitivity substrates.

What controls should be used when working with CAMTA1 antibodies?

Rigorous control selection is essential for validating results obtained with CAMTA1 antibodies:

• Positive Controls:

  • For Western blotting: Lysates from brain tissue or cell lines with known CAMTA1 expression

  • For IHC/IF: Brain tissue sections or confirmed EHE cases

  • Recombinant CAMTA1 protein can serve as a positive control for antibody validation

• Negative Controls:

  • For WB: Lysates from cell lines with CAMTA1 knockdown or tissues not expressing CAMTA1

  • For IHC/IF: Non-EHE epithelioid mesenchymal neoplasms, which consistently show negative staining

  • Omission of primary antibody to control for non-specific binding of secondary antibodies

• Specificity Controls:

  • Peptide competition assays using the immunizing peptide to confirm antibody specificity

  • Parallel testing with multiple CAMTA1 antibodies targeting different epitopes

  • siRNA knockdown experiments to confirm antibody specificity in cell culture systems

What are common issues in CAMTA1 detection and how can they be resolved?

Researchers may encounter several challenges when working with CAMTA1 antibodies:

• High Background Signal:

  • Problem: Non-specific binding leading to high background, particularly in IHC and IF

  • Solution: Increase blocking time, optimize antibody dilution, and use more stringent washing conditions. Consider using different blocking agents (BSA vs. milk) and adding 0.1-0.3% Triton X-100 to washing buffers for IHC/IF applications.

• Weak or Absent Signal:

  • Problem: Insufficient epitope exposure or low antibody sensitivity

  • Solution: For IHC, optimize antigen retrieval methods (heat-induced epitope retrieval with citrate or EDTA buffers). For WB, ensure complete protein denaturation and consider longer exposure times or more sensitive detection systems.

• Inconsistent Results Between Experiments:

  • Problem: Variation in staining intensity or pattern between experiments

  • Solution: Standardize all experimental conditions, including fixation time, antibody incubation temperature and duration, and detection reagents. Include consistent positive and negative controls in each experiment.

• Multiple Bands in Western Blotting:

  • Problem: Detection of multiple bands beyond the expected 183 kDa CAMTA1 protein

  • Solution: Verify whether smaller bands represent degradation products, isoforms, or non-specific binding. Use freshly prepared samples with protease inhibitors and optimize blocking conditions.

How can specificity of CAMTA1 antibodies be validated?

Validating the specificity of CAMTA1 antibodies is crucial for ensuring reliable research results:

• Peptide Competition Assays: Pre-incubate the CAMTA1 antibody with its immunizing peptide before application to the sample. Specific binding should be blocked by the peptide, resulting in signal reduction or elimination.

• Multiple Antibody Approach: Use antibodies from different sources or those targeting different CAMTA1 epitopes and compare staining patterns. Consistent results across different antibodies increase confidence in specificity.

• Genetic Manipulation: Perform knockdown or knockout of CAMTA1 using siRNA, shRNA, or CRISPR-Cas9 in appropriate cell lines, then confirm reduced or absent antibody binding.

• Correlation with mRNA Expression: Correlate protein detection with mRNA levels determined by qPCR or RNA-seq to ensure concordance between transcript and protein expression patterns.

• Mass Spectrometry Validation: For definitive validation, perform immunoprecipitation with the CAMTA1 antibody followed by mass spectrometry to confirm the identity of the precipitated protein.

What are the considerations for using CAMTA1 antibodies across different species?

When using CAMTA1 antibodies across different species, researchers should consider:

• Sequence Homology: Verify the degree of sequence conservation between species in the region recognized by the antibody. Higher homology increases the likelihood of cross-reactivity.

• Validated Reactivity: Check manufacturer specifications for validated species reactivity. Many CAMTA1 antibodies are validated for both human and mouse samples, but reactivity with other species may vary .

• Empirical Testing: Even with predicted cross-reactivity, empirical validation is essential. Test the antibody on positive control samples from each species of interest before proceeding with experimental samples.

• Optimization for Each Species: Antibody dilutions and detection protocols optimized for one species may not be optimal for another. Perform separate optimization for each species, adjusting dilutions and incubation conditions as needed.

• Species-Specific Controls: Always include species-appropriate positive and negative controls to validate antibody performance in each experimental system.

How can researchers quantify CAMTA1 expression levels accurately?

Accurate quantification of CAMTA1 expression requires appropriate methodological approaches:

• Western Blot Quantification:

  • Use internal loading controls (β-actin, GAPDH) for normalization

  • Ensure detection is in the linear range of the assay by performing dilution series

  • Use densitometry software with background subtraction capabilities

  • Include a standard curve of recombinant protein when absolute quantification is needed

• Immunohistochemistry Quantification:

  • Develop a consistent scoring system based on staining intensity and percentage of positive cells

  • Consider automated image analysis systems for more objective quantification

  • Use tissue microarrays with control samples for standardization across multiple specimens

• ELISA-Based Quantification:

  • Follow manufacturer's recommended dilutions (1:5000-1:20000)

  • Include standard curves with recombinant CAMTA1 protein

  • Ensure samples are within the dynamic range of the assay

• Flow Cytometry:

  • For cellular quantification of CAMTA1, optimize permeabilization protocols for nuclear proteins

  • Use appropriate isotype controls and fluorescence-minus-one (FMO) controls

  • Quantify using median fluorescence intensity (MFI) rather than percent positive cells

How does CAMTA1 expression relate to tumor biology?

CAMTA1 has emerging significance in tumor biology, with evidence suggesting tumor suppressor activity:

Experimental studies have demonstrated that induction of CAMTA1 in neuroblastoma SH-EP cells significantly decreases colony formation ability and growth rate . Cell cycle analysis reveals that CAMTA1 induction leads to G1/G0 arrest, with a significant increase in the proportion of cells in G1/G0 phase (from 59.3% to 69.9%) and a corresponding decrease in S-phase cells (from 29.2% to 19.5%) .

These findings suggest that CAMTA1 may function as a tumor suppressor by regulating cell cycle progression and inhibiting proliferation. This aligns with its chromosomal location at 1p36, a region frequently deleted in various human cancers .

In contrast, the WWTR1-CAMTA1 fusion gene resulting from t(1;3)(p36.3;q25) translocation leads to aberrant expression and activity, contributing to the pathogenesis of epithelioid hemangioendothelioma. This suggests that the fusion protein may have gained oncogenic properties distinct from wild-type CAMTA1 .

What is known about the WWTR1-CAMTA1 fusion gene in disease pathology?

The WWTR1-CAMTA1 fusion gene represents a defining genetic alteration in epithelioid hemangioendothelioma (EHE):

Approximately 90% of EHE cases harbor the WWTR1-CAMTA1 fusion gene resulting from recurrent translocations involving chromosomal regions 1p36.3 and 3q25 . This fusion gene leads to overexpression of both component proteins, with the fusion protein likely retaining functional domains from both partners .

The fusion creates a unique molecular entity where WWTR1 (widely expressed across many cell types) is joined with CAMTA1 (normally restricted to brain tissue). This results in aberrant expression of CAMTA1 domains in endothelial cells, potentially driving neoplastic transformation through dysregulated transcriptional programs .

The remaining EHE cases (<5%) that lack the WWTR1-CAMTA1 fusion typically harbor an alternative YAP1-TFE3 fusion gene. These distinct genetic subgroups may represent clinically relevant disease subtypes with potentially different prognoses or therapeutic vulnerabilities .

What are emerging applications for CAMTA1 antibodies in cancer research?

CAMTA1 antibodies are finding expanding applications in cancer research:

• Diagnostic Biomarker Development: Nuclear CAMTA1 expression has proven highly specific for EHE, distinguishing it from histologic mimics including benign epithelioid vascular tumors, epithelioid angiosarcoma, and epithelioid sarcoma . This diagnostic utility could be expanded to develop standardized immunohistochemical panels for difficult-to-diagnose vascular neoplasms.

• Prognostic Marker Investigation: Differences in CAMTA1 positivity between conventional (92%) and "malignant" (64%) EHE histologies suggest potential associations with tumor behavior that merit further investigation . Researchers could explore whether CAMTA1 staining intensity or pattern correlates with clinical outcomes.

• Therapeutic Target Identification: Understanding the functional consequences of WWTR1-CAMTA1 fusion may reveal targetable dependencies in EHE. Antibodies can facilitate mechanistic studies to identify potential therapeutic vulnerabilities.

• Liquid Biopsy Development: Exploring whether circulating tumor cells or cell-free DNA from EHE patients can be detected using CAMTA1 antibodies could lead to minimally invasive diagnostic or monitoring approaches.

How might CAMTA1 immunohistochemistry be integrated into diagnostic algorithms?

CAMTA1 immunohistochemistry has significant potential for integration into diagnostic algorithms:

• Sequential Testing Approach: For suspected vascular neoplasms, CAMTA1 testing could be implemented after initial vascular marker confirmation (CD31, CD34, ERG) to specifically identify EHE cases. This stepwise approach conserves resources while maintaining diagnostic accuracy.

• Complementary Panel with TFE3: Given that some EHE cases are CAMTA1-negative but TFE3-positive, a combined testing approach could increase diagnostic sensitivity . Researchers have found that 6 of 8 CAMTA1-negative EHE cases demonstrated TFE3 positivity, suggesting that these markers are largely complementary.

• Correlation with Molecular Testing: In challenging cases, correlation between CAMTA1 immunohistochemistry and molecular confirmation of the WWTR1-CAMTA1 fusion (via FISH or RT-PCR) could provide definitive diagnosis. This integrated approach combines the efficiency of immunohistochemistry with the specificity of molecular testing.

• Standardized Reporting Criteria: Development of standardized criteria for interpreting and reporting CAMTA1 immunohistochemistry results would facilitate consistent application across pathology laboratories and ensure reliable diagnostic information for clinical decision-making.