CALML5 Antibody

What is CALML5 and why is it significant in research?

CALML5 (Calmodulin-like 5), also known as calmodulin-like skin protein (CLSP), is a 146 amino acid calcium-binding protein predominantly expressed in the epidermis. It contains four EF-hand domains critical for calcium binding, enabling it to function similarly to calmodulin as a calcium sensor that modulates cellular processes . CALML5 plays a significant role in keratinocyte differentiation and is restricted to differentiating keratinocytes. It associates with transglutaminase 3, a key enzyme in terminal differentiation of keratinocytes . Recent research has identified CALML5 as a potential biomarker in various cancers, including thymic carcinoma and cutaneous melanoma, making it an important target for antibody-based research .

What applications are CALML5 antibodies validated for?

CALML5 antibodies have been validated for multiple research applications:

Validation data from different antibodies confirms reactivity with human samples, with some antibodies also showing reactivity with rat and mouse samples depending on the specific antibody clone .

What tissue samples and cell types show optimal CALML5 expression for antibody testing?

CALML5 shows tissue-specific expression patterns that researchers should consider when designing experiments:

• High expression tissues: Reconstructed epidermis, differentiating keratinocytes, human skin cancer tissue, human breast cancer tissue

• Cancer tissues expressing CALML5: Thymic squamous cell carcinoma (73.1% of cases), human colorectal cancer, human ovarian cancer, cutaneous melanoma

• Cell lines: A431 (human epidermoid carcinoma cells) have been validated for CALML5 detection

When designing experiments, researchers should consider that CALML5 expression is primarily cytoplasmic and nuclear in cancer cells, with diffuse staining patterns making it easier to detect in smaller samples compared to membrane-bound markers .

How should I optimize antigen retrieval for CALML5 immunohistochemistry?

Successful CALML5 immunohistochemistry depends on proper antigen retrieval:

For formalin-fixed paraffin-embedded (FFPE) tissues, the following protocol is recommended:

• Primary retrieval method: Use TE buffer at pH 9.0 for optimal epitope exposure

• Alternative method: Citrate buffer at pH 6.0 may be used as an alternative, though potentially with reduced sensitivity

• Section thickness: Use 4 μm thick sections for optimal results

• Deparaffinization and hydration: Complete deparaffinization and proper hydration is critical before applying antibodies

When evaluating results, independent assessment by at least two observers without prior knowledge of clinicopathological data is recommended to ensure objective interpretation, as implemented in the thymic carcinoma differentiation study .

What controls should be included when validating CALML5 antibodies in new experimental systems?

Proper validation of CALML5 antibodies requires appropriate controls:

• Positive tissue controls:

  • Human skin samples (normal differentiated keratinocytes)

  • Known CALML5-positive cancer samples (thymic squamous cell carcinoma, skin cancer)

• Negative tissue controls:

  • Type B3 thymoma tissue (shown to be negative in 94.7% of cases)

  • Tissues with known low CALML5 expression

• Experimental controls:

  • Antibody titration: Following manufacturer recommendations to determine optimal concentration (e.g., dilution series from 1:50-1:1200)

  • Isotype controls: Using matching IgG isotype (e.g., Rabbit IgG or Mouse IgG2a κ depending on antibody)

  • Blocking peptide controls: When available, using corresponding blocking peptides to confirm specificity

• Advanced validation:

  • CALML5 knockdown or knockout cell lines to confirm antibody specificity

  • Western blot validation showing the expected 16 kDa band

The experimental setup should be titrated for each specific testing system to obtain optimal results, as emphasized in manufacturer guidelines .

How do different fixation methods affect CALML5 antibody performance?

Research indicates that fixation methods can significantly impact CALML5 antibody performance:

• Formalin fixation: Standard 10% neutral buffered formalin fixation is compatible with most CALML5 antibodies for IHC applications

• Paraformaldehyde fixation: Appropriate for IF applications with cultured cells

• Fresh-frozen tissue vs. FFPE: While most validation data is based on FFPE tissues, some antibodies may perform better on fresh-frozen sections to preserve certain epitopes

• Fixation time: Overfixation may mask epitopes and require more aggressive antigen retrieval, while underfixation may result in poor tissue morphology

For challenging samples, dual validation using both IHC and IF techniques can provide more reliable results. Additionally, confirming protein expression with Western blotting using fresh or frozen tissue lysates can validate antibody performance independent of fixation artifacts.

How reliable is CALML5 as a diagnostic marker for differentiating thymic carcinoma from thymoma?

CALML5 has emerged as a promising diagnostic marker for differentiating thymic squamous cell carcinoma from type B3 thymoma:

Key advantages of CALML5 over existing markers:

• Higher sensitivity than CD5 for thymic carcinoma detection

• Equal or higher specificity compared to c-kit

• Diffuse cytoplasmic distribution creates a larger staining area, making evaluation easier in small biopsy samples

• Some cases show CD5−/c-kit−/CALML5+ thymic carcinoma, indicating CALML5's value in otherwise marker-negative cases

Researchers should note that while no single marker offers perfect sensitivity and specificity, a panel approach incorporating CALML5 with traditional markers provides optimal diagnostic accuracy .

What is the relationship between CALML5 expression and cancer progression mechanisms?

Research has revealed complex relationships between CALML5 expression and cancer progression:

These findings suggest CALML5 may serve as both a diagnostic marker and potential therapeutic target in various cancers.

How does CALML5 interact with the lactylation pathway in cancer development?

Recent research has identified CALML5 as a core lactylation-associated gene in cutaneous melanoma (CM):

These findings suggest that targeting the lactylation pathway through CALML5 might represent a novel therapeutic approach for cancer treatment.

What are common causes of false positive or false negative results with CALML5 antibodies?

Researchers should be aware of several potential sources of errors when working with CALML5 antibodies:

Causes of false positives:

• Cross-reactivity: Some antibodies may cross-react with similar calcium-binding proteins, particularly other calmodulin family members

• Excessive antibody concentration: Using antibody concentrations above recommended dilution ranges (1:50-1:1200 for IHC depending on the specific antibody)

• Insufficient blocking: Inadequate blocking can lead to non-specific binding

• Endogenous peroxidase activity: Incomplete quenching of endogenous peroxidase activity in IHC

• Detection system issues: Overly sensitive detection systems or excessive substrate development time

Causes of false negatives:

• Epitope masking: Improper fixation or inadequate antigen retrieval (particularly important to use recommended TE buffer pH 9.0 or alternative citrate buffer pH 6.0)

• Antibody degradation: Improper storage (should be stored at -20°C; stable for one year after shipment)

• Insufficient antibody concentration: Using dilutions beyond the recommended range

• Sample issues: Prolonged fixation or improper tissue handling

• Detection sensitivity: Using detection systems with inadequate sensitivity

To minimize these issues, researchers should:

• Validate antibodies using known positive and negative controls

• Follow recommended protocols for antigen retrieval

• Optimize antibody concentration for each application and tissue type

• Consider using alternative antibody clones if persistent issues occur

How should CALML5 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of CALML5 antibodies is critical for maintaining their performance:

Storage conditions:

• Store at -20°C for long-term storage (typical shelf life of one year after shipment)

• For antibodies in glycerol buffer (e.g., PBS with 0.02% sodium azide and 50% glycerol pH 7.3), aliquoting is unnecessary for -20°C storage

• For antibodies without glycerol, aliquoting is recommended to avoid freeze-thaw cycles

Handling recommendations:

• Thaw antibodies completely before use and mix gently by inverting

• Avoid repeated freeze-thaw cycles that can lead to protein denaturation and loss of activity

• For short-term storage (1-2 weeks), antibodies can be kept at 4°C

• When diluting antibodies, use recommended buffers (typically PBS with stabilizers)

• For preservative-free antibodies, it is recommended to add sodium azide (final concentration 0.05%-0.1%) to avoid contamination

Stability considerations:

• Antibody conjugates (HRP, FITC, PE, etc.) may have different stability profiles than unconjugated antibodies

• Conjugated antibodies should be protected from light to prevent photobleaching

• Some small antibody aliquots (e.g., 20μl sizes) may contain 0.1% BSA as an additional stabilizer

Following these recommendations will help ensure consistent and reproducible results across experiments.

How can I validate specificity when comparing results from different CALML5 antibody clones?

When comparing results from different CALML5 antibody clones, researchers should implement a systematic validation approach:

• Epitope comparison:

  • Compare binding regions of different antibodies (e.g., AA 1-146, AA 2-146, AA 47-146, or C-terminal regions)

  • Antibodies targeting different regions may yield different staining patterns

• Cross-validation methods:

  • Western blot: Confirm a single band at expected 16 kDa molecular weight

  • IHC on serial sections: Compare staining patterns of different antibodies on consecutive tissue sections

  • IF co-localization: Use different antibody clones with distinct fluorescent labels to verify co-localization

  • siRNA/CRISPR knockdown validation: Verify signal reduction with all antibody clones in knockdown models

• Isotype and host considerations:

  • Different isotypes (e.g., IgG2a κ vs. polyclonal IgG) may have different non-specific binding properties

  • Host species (rabbit vs. mouse) may affect background in certain tissues

• Quantitative comparison:

  • Standardize detection methods (same secondary antibodies/detection systems)

  • Use digital image analysis to quantify staining intensity and distribution

  • Compare results with mRNA expression data from methods like CAGE sequencing

• Documentation of differences:

  • Create a comparison table documenting performance of each antibody across applications

  • Note sensitivity, specificity, and optimal conditions for each clone

This systematic approach ensures that observed differences in results are attributable to genuine biological variation rather than technical artifacts from different antibody characteristics.

What novel applications are being developed for CALML5 antibodies beyond traditional cancer diagnostics?

Research with CALML5 antibodies is expanding beyond traditional cancer diagnostics into several promising areas:

• Immune checkpoint therapy selection:

  • CALML5 expression correlates with immune checkpoint molecules in cutaneous melanoma

  • This correlation could potentially identify patients likely to respond to immune checkpoint inhibitors

• Skin barrier research:

  • Given CALML5's role in keratinocyte differentiation and interaction with transglutaminase 3

  • CALML5 antibodies may help investigate skin barrier dysfunction in conditions like atopic dermatitis or psoriasis

• Calcium signaling research:

  • As a calcium-binding protein with four EF-hand domains

  • CALML5 antibodies can help study tissue-specific calcium signaling pathways

• Liquid biopsy development:

  • Potential use in detecting circulating tumor cells or extracellular vesicles expressing CALML5

  • Development of highly sensitive detection methods for early cancer diagnosis

• Therapy response monitoring:

  • CALML5 overexpression increases cisplatin sensitivity in cancer cells

  • Antibodies could help monitor treatment response and resistance development

• Combination with lactylation markers:

  • Given CALML5's identification as a lactylation-associated gene

  • Combined detection of CALML5 and lactylation modifications could provide insights into cancer metabolism

These emerging applications suggest CALML5 antibodies have potential beyond their current diagnostic applications in cancer research.

How might CALML5 antibodies contribute to understanding the tumor immune microenvironment?

Recent research has uncovered significant relationships between CALML5 and the tumor immune microenvironment:

• Immune cell infiltration correlations:
  Studies have shown that CALML5 expression in cutaneous melanoma positively correlates with infiltration of:

  • Activated/resting dendritic cells

  • Resting mast cells

  • Activated NK cells

  • Neutrophils

  • Regulatory T cells

• Immune pathway associations:
  CALML5 has been found to be enriched in immune-associated pathways including:

  • T cell receptor signaling pathway

  • Natural killer cell mediated cytotoxicity

  • Other immune-related signaling pathways

• Immune checkpoint relationship:
  In high-risk cutaneous melanoma patients:

  • Decreased expression of numerous immune checkpoint molecules

  • Exceptions include increased expression of TNFRSF14, CD276, VTCN1, and TNFSF9

  • These molecules play roles in immune evasion mechanisms

• Lactylation connection to immune modulation:

  • Lactylation is connected with immune regulation

  • CALML5 as a core lactylation-associated gene potentially links metabolism to immune response

• Potential for immunotherapy stratification:

  • CALML5 expression patterns might help identify patients more likely to benefit from specific immunotherapies

  • Could serve as part of a biomarker panel for immunotherapy selection

These findings suggest CALML5 antibodies could become valuable tools for studying the complex interplay between cancer cells and the immune microenvironment, potentially guiding personalized immunotherapy approaches.

What role might CALML5 antibodies play in multi-marker diagnostic panels?

CALML5 antibodies show significant promise as components of multi-marker diagnostic panels:

When designing such panels, researchers should consider standardized protocols that accommodate the optimal conditions for each antibody and marker, as well as appropriate multiplexing strategies to maximize information from limited tissue samples.