CTTN Antibody

What is the optimal fixation method for CTTN detection in immunohistochemistry?

For optimal CTTN detection in paraffin-embedded tissues, heat-induced epitope retrieval using basic antigen retrieval reagents is recommended. Most protocols employ immersion fixation followed by overnight incubation with the primary antibody at 4°C using concentrations between 5-15 μg/mL. For example, when studying CTTN in human prostate tissue, researchers successfully used heat-induced epitope retrieval with Anti-Mouse HRP-DAB detection systems, which revealed specific localization to plasma membranes of glandular epithelial cells . For phosphorylated CTTN detection (particularly at Y460), standard paraffin embedding protocols yield good results with human breast carcinoma tissues .

Which CTTN antibody is most suitable for detecting expression in multiple species simultaneously?

For multi-species detection, researchers should select antibodies validated across the target species. The Anti-Cortactin Rabbit Monoclonal Antibody (M01253) demonstrates reactivity with human, mouse, and rat samples across multiple applications (WB, IHC, ICC/IF, IP, Flow Cytometry) . Similarly, the 4F11 clone has been validated for recognizing CTTN in avian, rat, mouse, human, bovine, and hamster tissues . When considering cross-reactivity for species not explicitly tested, sequence homology analysis is recommended, as demonstrated in a customer inquiry about pig tissue reactivity with the M01253 antibody .

How do phospho-specific CTTN antibodies differ in their utility for studying cytoskeletal dynamics?

Phospho-specific CTTN antibodies target distinct phosphorylation sites that regulate different aspects of cortactin function in cytoskeletal dynamics. Phosphorylation at Tyr421, Tyr466, and Tyr482/486 (mouse/human) critically regulates cortactin's cytoskeletal activity in endothelial cells . When investigating barrier function and vascular permeability, phospho-Y460 antibodies are particularly valuable as this site is proximal to the functionally critical Tyr486 residue involved in endothelial cell motility and cytoskeletal rearrangement . Researchers should select phospho-specific antibodies based on the specific pathway under investigation:

• pTyr421 antibodies: Useful for studying initial phosphorylation events in SRC-mediated pathways

• pTyr466/pTyr460 antibodies: Valuable for investigating actin polymerization dynamics and barrier function

• pTyr486 antibodies: Critical for examining lamellipodia formation and cell migration

For proper interpretation, researchers should correlate phosphorylation patterns with functional outcomes using complementary techniques such as live cell imaging or biophysical measurements of cellular responses.

What methodological considerations should be addressed when using CTTN antibodies for studying the functional impact of CTTN SNPs?

When investigating the functional impact of CTTN SNPs such as S484N (rs56162978), researchers should implement a comprehensive methodological approach:

• Genotyping verification: Confirm the SNP presence using appropriate PCR primers flanking the nucleotide substitution site (e.g., 5'-TAGGCACATTTGGGGCATCG-3' and 5'-CGAACATCAAGGCATCTGTGC-3')

• Expression system selection: Consider using CRISPR/Cas9 to introduce homozygous amino acid substitutions in relevant cells like human aorta endothelial cells (teloHAECs)

• Functional assays: Implement multiple complementary approaches:

  • Trans-endothelial electrical resistance (TER) measurements to assess barrier function

  • Biophysical studies of lamellipodia dynamics

  • Protein-protein interaction assays to examine binding to critical partners (e.g., nmMLCK)

  • Phosphorylation status analysis at nearby critical sites (e.g., Tyr486)

• In vivo validation: Consider heterozygous models (e.g., Cttn+/− mice) combined with lung injury models (LPS or ventilator-induced) to assess physiological impact through measurements of:

  • Bronchoalveolar lavage (BAL) protein levels

  • Inflammatory cell counts

  • Histological examination

The S484N SNP has been specifically associated with increased lung vascular permeability and inflammatory injury in Black patients, which was verified using these methodological approaches .

How can researchers resolve inconsistent CTTN antibody staining patterns between Western blot and immunohistochemistry results?

Inconsistencies between Western blot and immunohistochemistry results for CTTN may arise from several factors:

• Epitope accessibility: CTTN's complex structure and interactions with cytoskeletal components may mask epitopes differently in native versus denatured states. For Western blots, ensure complete denaturation using appropriate buffers containing SDS and reducing agents.

• Isoform specificity: CTTN has multiple isoforms (with expected protein mass of 61.6 kDa, but detected at 80-85 kDa due to post-translational modifications) . When optimizing Western blots, researchers should note that clone 4F11 recognizes "80 kDa and 85 kDa CTTN in avian cells, and 80 kDa protein in rodent and human cells" .

• Tissue-specific expression patterns: CTTN is expressed differentially across tissues, including "right ovary, mammary gland, placenta, testis, leukemic t-cell, cervix carcinoma, prostate cancer, platelet, cervix carcinoma erythroleukemia, liver" . Confirm antibody validation in your specific tissue type.

• Fixation and retrieval methods: For IHC, optimize antigen retrieval conditions. Several researchers reported success with heat-induced epitope retrieval using basic buffers for CTTN detection in paraffin-embedded tissues .

If discrepancies persist, validate results using an alternative antibody clone targeting a different epitope or implement orthogonal validation techniques such as RNA expression analysis.

What considerations should be made when selecting storage conditions for CTTN antibodies to maintain optimal reactivity?

Proper storage of CTTN antibodies is critical for maintaining reactivity and specificity:

• Long-term storage: Store at -20°C for up to one year in aliquots to minimize freeze-thaw cycles

• Short-term storage: For frequent use within one month, 4°C storage is acceptable

• Buffer considerations: Most CTTN antibodies are supplied in PBS with preservatives (e.g., 0.05% sodium azide) . Avoid buffer exchanges unless necessary as this may impact stability

• Freeze-thaw cycles: Minimize repeated freeze-thaw cycles as they significantly reduce antibody performance. As noted in the M01253 product information: "Avoid repeated freeze-thaw cycles"

• Shipping conditions: Note that antibodies shipped on cold packs should be immediately transferred to appropriate storage upon receipt

For critical experiments, researchers should validate antibody performance after extended storage by testing with positive control samples (e.g., cell lines known to express high levels of CTTN such as breast cancer or prostate cancer cell lines).

How should researchers interpret CTTN expression patterns in cancer tissues versus normal tissues?

When interpreting CTTN expression patterns in cancer versus normal tissues, researchers should consider:

• Baseline tissue expression: CTTN is normally expressed in multiple tissues including mammary gland, ovary, placenta, testis, and liver . Establish appropriate baseline controls from normal tissues.

• Subcellular localization: In normal tissues, CTTN typically localizes to cytoplasm and cytoskeleton, particularly at plasma membranes of glandular epithelial cells . In cancer cells, altered localization (especially to invadopodia) may be functionally significant.

• Amplification patterns: CTTN gene amplification occurs in certain tumors , suggesting a role in tumorigenesis. Quantitative analysis should compare expression levels normalized to appropriate housekeeping proteins.

• Phosphorylation status: Cancer-associated signaling often increases CTTN phosphorylation. Using phospho-specific antibodies targeting Tyr421, Tyr466, or Tyr486 provides insight into activation status rather than just expression level .

• Correlation with functional outcomes: Interpret CTTN expression in context with invasion markers, metastatic potential, and patient outcomes. High CTTN expression in breast cancer and squamous cell carcinomas of the head and neck correlates with invasive phenotypes .

For comprehensive analysis, combine IHC with other techniques like qPCR for mRNA expression and functional assays to assess migration/invasion capacity.

What controls are essential for validating phospho-specific CTTN antibody specificity in experimental systems?

For rigorous validation of phospho-specific CTTN antibody specificity, implement these essential controls:

• Phosphatase treatment control: Treat duplicate samples with lambda phosphatase to demonstrate signal reduction with phospho-specific antibodies

• Stimulation controls: Include samples from cells treated with:

  • SRC kinase activators (which phosphorylate CTTN at Tyr421, Tyr466, and Tyr482/486)

  • Actin cytoskeleton disruptors (cytochalasin D)

  • Barrier function modulators (thrombin for disruption, S1P for enhancement)

• Site-directed mutagenesis controls: Generate phospho-deficient (Y→F) and phospho-mimetic (Y→E) mutants of the target phosphorylation site

• Peptide competition: Pre-incubate antibody with phosphorylated and non-phosphorylated peptides corresponding to the target site

• Knockout/knockdown validation: Include samples from CTTN CRISPR knockout or siRNA knockdown cells as negative controls

For phospho-Y460 CTTN antibodies specifically, researchers should note this site's proximity to the functionally critical Tyr486 site involved in endothelial cell motility , and include controls addressing possible cross-reactivity between these closely positioned epitopes.

How can CTTN antibodies be utilized in the study of endothelial barrier dysfunction in acute respiratory distress syndrome (ARDS)?

CTTN antibodies offer valuable tools for investigating endothelial barrier dysfunction in ARDS through several methodological approaches:

• Genetic association studies:

  • Use CTTN antibodies to phenotype expression variations in patient samples with known CTTN SNPs

  • The S484N (rs56162978) SNP significantly associates with outcomes in sepsis mortality and acute chest syndrome severity in Black patients

• Ex vivo tissue analysis:

  • Compare phosphorylated CTTN levels in lung tissues from ARDS patients versus controls

  • Correlate with severity markers and clinical outcomes

• In vitro barrier models:

  • Implement transendothelial electrical resistance (TER) measurements in cells expressing wild-type versus mutant CTTN (S484N)

  • Monitor real-time responses to barrier disruptors (LPS, thrombin) and enhancers

• CTTN-nmMLCK interaction studies:

  • Use phospho-specific antibodies to track CTTN-Y486 phosphorylation levels

  • Assess binding to critical cytoskeletal regulators like non-muscle myosin light chain kinase (nmMLCK)

• In vivo models:

  • Utilize Cttn+/− heterozygous mice exposed to LPS or ventilator-induced lung injury

  • Quantify bronchoalveolar lavage protein levels (1808 ± 142 μg/ml in Cttn+/− vs. 1316 ± 200 μg/ml in WT with LPS treatment)

  • Measure neutrophil recruitment (6.60 ± 0.90 × 10^6 in Cttn+/− vs. 3.19 ± 0.50 × 10^6 in WT mice)

This multifaceted approach leverages CTTN antibodies to elucidate molecular mechanisms underlying vascular leak in ARDS, potentially identifying novel therapeutic targets.

What are the considerations for using CTTN antibodies in designing multi-specific antibody therapeutics?

The development of multi-specific antibody therapeutics incorporating anti-CTTN functionality requires several methodological considerations:

• Epitope selection:

  • Target functionally critical domains of CTTN that regulate cytoskeletal dynamics

  • Consider epitopes that are accessible in the native protein conformation

  • Avoid regions with high sequence variability across species if broad application is desired

• Antibody format design:

  • For trispecific constructs, carefully consider orientation and flexibility between binding domains

  • Evaluate binding kinetics using biolayer interferometry (BLI) with immobilized CTTN protein (50 μg/mL) and antibody flow rates of approximately 30 μL/minute

  • Determine association (kon) and dissociation (koff) rates to calculate KD values

• Functional validation:

  • Verify maintained binding activity of each domain in the multi-specific construct

  • Test against different subtypes of target antigens to ensure broad reactivity

  • Implement cell-based assays to confirm penetration and target engagement

• In vivo efficacy assessment:

  • Evaluate pharmacokinetics and tissue distribution

  • Use appropriate disease models (e.g., Cttn+/− mice with ventilator-induced lung injury)

  • Assess therapeutic delivery methods (e.g., liposomal delivery of CTTN transgenes)

• Specificity considerations:

  • Exclude cross-reactivity with related proteins

  • Confirm maintained specificity in the multi-specific format