COTL1 Antibody

What is COTL1 and what is its significance in research?

COTL1 (coactosin-like 1) is a 16 kDa protein composed of 142 amino acid residues that binds to F-actin in a calcium-independent manner. It shares similarities with Dictyostelium discoideum coactosin and is encoded by a gene located on chromosome 16q24.1 . COTL1 functions as an F-actin binding protein and has been identified as a 5-lipoxygenase (5LO) binding partner, potentially playing a role in leukotriene biosynthesis in leukocytes . Recent research has highlighted COTL1's significance in various pathological conditions, particularly its association with autoimmune disorders like rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), as well as its potential role in cancer progression and immune response modulation .

What applications are COTL1 antibodies typically used for?

COTL1 antibodies have been validated for multiple research applications:

These applications enable researchers to study COTL1 expression, localization, and interactions in various experimental contexts .

What is the expected molecular weight of COTL1 in Western blot analysis?

The calculated molecular weight of COTL1 is 16 kDa, which is typically observed in Western blot analysis. Some antibodies detect COTL1 in the 14-16 kDa range, consistent with the protein's expected size . When interpreting Western blot results, researchers should be aware that post-translational modifications may occasionally result in slight variations in the observed molecular weight .

What are the key differences between monoclonal and polyclonal COTL1 antibodies?

Selecting between monoclonal and polyclonal antibodies depends on the experimental requirements, with polyclonals offering broader epitope recognition and monoclonals providing higher specificity .

How can COTL1 antibodies be used to investigate immune cell infiltration in cancer?

Recent research has demonstrated that COTL1 has significant correlations with immunological checkpoints and immune infiltration cells in various cancer types . When designing experiments to investigate this:

• Use COTL1 antibodies for immunohistochemistry or immunofluorescence to assess COTL1 expression in tumor microenvironment

• Perform co-staining with immune cell markers (e.g., CD8, PD-L1) to evaluate correlation with COTL1 expression

• Compare COTL1 expression between tumor and adjacent normal tissues

• Correlate COTL1 expression with clinical parameters and survival data

Research has shown positive correlations between COTL1 expression, CD8, and PD-L1 in low-grade glioma (LGG), with high COTL1 expression associated with decreased patient survival . This suggests COTL1 may serve as an immunological and prognostic biomarker with potential implications for developing novel cancer therapies .

What methodologies are optimal for studying COTL1 polymorphisms in autoimmune disorders?

COTL1 polymorphisms have been associated with autoimmune disorders like RA and SLE. The optimal methodology for studying these associations involves:

• Sample collection and DNA extraction: From patients with RA or SLE and healthy controls

• Genotyping: For key COTL1 SNPs including c.-1124G>T, c.484G>A, c.588C>T, and c.1050T>A

• Clinical correlation: Analyzing relationship between genotypes and clinical parameters:

  • Anti-CCP antibody levels in RA patients (c.484G>A polymorphism has shown significant association)

  • Rheumatoid factor (RF) in RA patients

  • Anti-nuclear antibodies (ANA) in SLE patients

• Haplotype analysis: Comparing haplotype frequencies between patients and controls

• Protein expression analysis: Using COTL1 antibodies to compare protein expression levels between different genotypes

The c.484G>A polymorphism has shown significant association with anti-CCP antibody levels in RA patients (P = 0.03), suggesting that COTL1 polymorphisms might influence disease pathophysiology .

How can researchers investigate COTL1's interaction with 5-lipoxygenase?

COTL1 has been identified as a 5-lipoxygenase (5LO) binding partner through yeast two-hybrid screening, with the LKKAET-like motif of COTL1 interacting with 5LO involved in leukotriene biosynthesis in leukocytes . To study this interaction:

• Co-immunoprecipitation (Co-IP): Use COTL1 antibodies to pull down protein complexes, then probe for 5LO

• Reciprocal Co-IP: Use 5LO antibodies for immunoprecipitation, then detect COTL1

• Proximity ligation assay (PLA): To visualize and quantify COTL1-5LO interactions in situ

• Mutagenesis studies: To confirm the role of the LKKAET-like motif in the interaction

• Functional assays: To assess how this interaction affects leukotriene production

This methodology allows researchers to explore how COTL1 may regulate inflammatory processes through its interaction with the leukotriene biosynthesis pathway.

What approaches are recommended for investigating COTL1's role in cancer stemness?

Research has shown that COTL1 is associated with DNA and RNA stemness in numerous tumor types, suggesting a potential role in cancer stem cell biology . To investigate this:

• Expression analysis: Use COTL1 antibodies to compare expression between cancer stem cells and differentiated tumor cells

• Correlation studies: Analyze relationship between COTL1 expression and established stemness markers

• Knockdown/overexpression experiments: Evaluate effects on:

  • Self-renewal (sphere formation assays)

  • Expression of stemness genes

  • Tumor-initiating capacity in vivo

• ChIP-seq analysis: To identify potential regulatory mechanisms controlling COTL1 expression in stem-like cells

• Patient-derived xenograft models: To assess how COTL1 expression correlates with tumor initiation and propagation

This multi-faceted approach can help elucidate COTL1's role in maintaining cancer stem cell properties and potentially identify new therapeutic targets.

What are the optimal sample preparation and antigen retrieval methods for COTL1 immunohistochemistry?

For optimal COTL1 detection in IHC applications:

Both monoclonal and polyclonal COTL1 antibodies have been successfully used for IHC applications, with the choice depending on specific experimental requirements .

How can non-specific binding be minimized when using COTL1 antibodies?

To minimize non-specific binding and optimize signal-to-noise ratio:

• Antibody titration: Determine optimal concentration through serial dilutions (recommended ranges: 1:2000-1:16000 for WB, 1:50-1:500 for IHC)

• Blocking optimization: Use 5% BSA or 5% non-fat milk in TBS-T for Western blot and 5-10% normal serum from the secondary antibody host species for IHC/IF

• Wash protocol optimization: Increase number and duration of washes

• Secondary antibody selection: Choose highly cross-adsorbed secondaries appropriate for your experimental system

• Negative controls: Include no-primary-antibody controls and ideally COTL1 knockout/knockdown samples

• Positive controls: Include samples known to express COTL1 (e.g., A549 cells, HeLa cells, human peripheral blood platelets)

These approaches help ensure that signals detected represent genuine COTL1 expression rather than artifacts.

What controls should be included when performing COTL1 Western blot analysis?

For robust and interpretable Western blot experiments:

• Positive controls: Cell lines with confirmed COTL1 expression (A549, HeLa, A431 cells)

• Loading controls:

  • For cytoplasmic proteins: GAPDH, β-actin

  • For total protein normalization: Stain-free technology or total protein stains

• Molecular weight markers: To confirm the observed band matches the expected 16 kDa size of COTL1

• Knockdown/knockout validation: Where possible, include COTL1 knockdown samples to confirm antibody specificity

• Recombinant protein: Pure COTL1 protein can serve as a positive control and sizing reference

Including these controls ensures experimental validity and aids in troubleshooting if unexpected results are obtained.

What are key considerations for immunoprecipitation experiments using COTL1 antibodies?

For successful COTL1 immunoprecipitation:

• Antibody selection: Use antibodies validated for IP applications (e.g., 10781-1-AP at 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate)

• Lysis buffer optimization: Use buffers that preserve protein-protein interactions while efficiently extracting COTL1

• Pre-clearing lysates: To reduce non-specific binding

• Bead selection: Protein A/G beads for rabbit polyclonal antibodies, Protein G for mouse monoclonals

• Controls:

  • IgG control (same species as COTL1 antibody)

  • Input sample (pre-IP lysate)

  • Non-bound fraction

• Elution conditions: Optimize to maintain integrity of co-precipitated proteins

• Detection method: Western blot with a different COTL1 antibody (if available) to avoid detecting the IP antibody

IP experiments have successfully detected COTL1 in mouse kidney tissue, demonstrating the feasibility of this approach .

How should researchers interpret COTL1 expression data in cancer studies?

When analyzing COTL1 expression in cancer contexts:

• Expression patterns: COTL1 is highly expressed in most cancers compared to normal tissues

• Prognostic significance: High expression correlates with decreased survival in glioma, glioblastoma multiforme, and pan-kidney cohorts

• Correlation analysis: Examine relationships with:

  • Tumor mutation burden (TMB)

  • Microsatellite instability (MSI)

  • Neoantigen (NEO) load

  • PD-L1 expression

• Genomic instability markers: COTL1 shows favorable relationships with:

  • Loss of heterozygosity (LOH)

  • Homologous recombination deficiency (HRD)

  • Mutant allele tumor heterogeneity (MATH)

These analyses can help determine whether COTL1 may serve as a useful biomarker for patient stratification, prognosis, or potential therapeutic targeting .

What methodological approaches are recommended for studying COTL1 in proteomics research?

For proteomic investigation of COTL1:

• Two-dimensional electrophoresis (2-DE): Has been successfully used to identify differential expression of COTL1 in disease states

• Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS): For protein identification and characterization

• Sample types: PBMCs, plasma, synovial fluid have all been used successfully

• Validation approaches:

  • Western blot with COTL1 antibodies

  • Immunohistochemistry on relevant tissues

  • qPCR for mRNA expression correlation

• Comparative analysis: Between disease (RA, SLE, cancer) and healthy control samples

These proteomic approaches have identified COTL1 as differentially expressed in several pathological conditions, leading to further genetic and functional studies .

How can researchers correlate COTL1 genotypes with protein expression and function?

To establish relationships between COTL1 genotypes, protein expression, and function:

• Genotyping: Determine COTL1 polymorphisms (c.-1124G>T, c.484G>A, etc.) in study populations

• Protein quantification:

  • Western blot with COTL1 antibodies

  • ELISA for serum/plasma COTL1 levels

  • Immunohistochemistry for tissue expression patterns

• Functional assays:

  • F-actin binding capacity

  • 5-lipoxygenase interaction studies

  • Cell migration and invasion assays

• Statistical analysis:

  • ANOVA or t-tests for comparing protein levels between genotypes

  • Correlation analysis between protein levels and functional outcomes

  • Multivariate analysis to control for confounding factors

Research has shown that COTL1 polymorphisms (particularly c.484G>A) correlate with anti-CCP antibody levels in RA patients, suggesting functional consequences of genetic variation .

What experimental designs are appropriate for validating COTL1 as a biomarker?

To validate COTL1 as a biomarker for diseases like cancer or autoimmune disorders:

• Discovery phase:

  • Pan-cancer analysis of COTL1 expression (already shows potential in multiple cancer types)

  • Proteomic identification of differential expression (as seen in RA patients)

• Validation phase:

  • Independent patient cohorts

  • Multiple detection methods (IHC, WB, ELISA)

  • Correlation with established biomarkers

• Clinical correlation:

  • Survival analysis stratified by COTL1 expression

  • Response to therapy prediction

  • Correlation with disease activity measures

• Functional validation:

  • In vitro studies on mechanisms

  • Animal models when applicable

• Assay development:

  • Standardization of antibody-based detection methods

  • Determination of clinically relevant cutoff values

Following this structured approach can help establish whether COTL1 has genuine utility as a biomarker for specific conditions and clinical applications.

How might COTL1 serve as a target for immunotherapeutic approaches?

COTL1 shows promising characteristics for immunotherapy development:

• Association with immune checkpoints: COTL1 shows positive correlations with immunological checkpoints in multiple cancer types

• Immune cell infiltration: Correlates with immune infiltration cells in tumor microenvironments

• Relationship with established immunotherapy targets: Links to tumor mutation burden (TMB), microsatellite instability (MSI), neoantigen (NEO), and PD-L1 expression

• Survival impact: High expression correlates with decreased patient survival in certain cancers

Research approaches might include:

• Developing antibodies targeting COTL1 for therapeutic applications

• Investigating combination approaches with established checkpoint inhibitors

• Exploring COTL1's role in regulating immune response to tumors

The positive correlation between COTL1 expression, CD8, and PD-L1 in low-grade glioma suggests potential synergistic approaches .

What is known about the role of COTL1 in actin cytoskeleton regulation?

COTL1 was identified as a filamentous actin (F-actin) binding protein that binds to F-actin in a calcium-independent manner . To investigate this function:

• Co-localization studies: Using COTL1 antibodies and F-actin markers

• Binding assays: To characterize the specific domains involved in F-actin interaction

• Functional impact:

  • Effects on actin polymerization/depolymerization

  • Influence on cell migration and invasion

  • Role in cellular processes requiring cytoskeletal remodeling

• Interactome analysis: Identifying other cytoskeletal proteins that interact with COTL1

Understanding COTL1's role in cytoskeleton regulation could provide insights into its functions in normal physiology and disease states, particularly in processes like immune cell migration and cancer metastasis.