FERMT1 Antibody

What is FERMT1 and why is it significant in cancer research?

FERMT1 (Fermitin family member 1) is a protein expressed in most epithelial tissues that primarily participates in cellular processes including cell adhesion, motility, and migration. Defects in this gene cause Kindler syndrome, a genetic disease characterized by fragile skin and increased risk of squamous cell carcinoma . FERMT1 has gained significant research interest due to its upregulation in multiple cancer types and association with poor prognosis.

FERMT1 has been demonstrated to play crucial roles in tumor proliferation, metastasis, and epithelial-mesenchymal transition (EMT). In nasopharyngeal carcinoma, FERMT1 knockdown significantly decreases cell proliferation, migration and invasion by mediating EMT and cell cycle arrest both in vitro and in vivo . Similarly, in non-small cell lung cancer, FERMT1 promotes migration and invasion through upregulation of plakophilin 3 and activation of the p38 MAPK signaling pathway . These findings make FERMT1 a valuable research target and potential therapeutic candidate in oncology.

What detection methods are available for FERMT1 in research applications?

Several methodological approaches can be employed for FERMT1 detection:

• Immunohistochemistry (IHC): FERMT1 antibodies have been effectively used for tissue staining with optimal dilutions around 1:200 for commercial antibodies such as those from Proteintech (22215-1-AP) . IHC allows visualization of FERMT1 expression patterns within tissue architecture and enables scoring systems for quantification (0 to 3+).

• Western blotting: For protein expression analysis in cell lysates or tissue homogenates. This method allows quantitative comparison of FERMT1 expression between experimental groups .

• ELISA: Sandwich enzyme immunoassays for quantitative measurement of FERMT1 in human serum, plasma, cell culture supernatants, and tissue homogenates. These assays typically have detection ranges of 78.13-5000 pg/mL with sensitivities below 39 pg/mL .

• Reverse transcription PCR (RT-PCR): For analysis of FERMT1 mRNA expression, allowing detection of transcriptional regulation .

How should I optimize FERMT1 antibody protocols for immunohistochemistry?

Optimizing IHC protocols for FERMT1 detection requires attention to several parameters:

Sample preparation:

• Formalin-fixed paraffin-embedded (FFPE) tissues should undergo appropriate antigen retrieval, typically heat-induced epitope retrieval in citrate buffer (pH 6.0)

• Optimal section thickness is 4-5 μm for consistent staining

Protocol optimization:

• Primary antibody concentration: Start with 1:200 dilution for commercial antibodies like Proteintech 22215-1-AP, then adjust based on signal-to-noise ratio

• Incubation time: Typically overnight at 4°C for primary antibody

• Detection system: Avidin-biotin complex (ABC) or polymer-based detection systems are suitable

• Counterstaining: Light hematoxylin counterstaining helps visualize tissue architecture

Scoring system:

• Implement a standardized scoring system (0, 1+, 2+, 3+) where:

  • 0 and 1+ indicate low expression

  • 2+ and 3+ indicate high expression

Controls:

• Always include positive control tissues (colon or lung carcinoma tissues are recommended)

• Include negative controls by omitting primary antibody

• Consider using FERMT1-knockout cell lines as biological negative controls

This methodological approach ensures reproducible results across experiments and between laboratories.

What are the recommended sample preparation techniques for FERMT1 protein detection?

Sample preparation is critical for accurate FERMT1 detection and varies by experimental method:

For tissue samples:

• Snap freeze tissues in liquid nitrogen immediately after collection

• Store at -80°C until processing

• For protein extraction, homogenize tissues in RIPA buffer supplemented with protease inhibitors

• Centrifuge at 12,000 g for 15 minutes at 4°C to remove debris

• Determine protein concentration using BCA or Bradford assay

For cell culture samples:

• Wash cells with cold PBS to remove media components

• Lyse cells directly in the culture plate using appropriate lysis buffer

• For suspension cells, pellet by centrifugation before lysis

• Process lysates as described for tissue samples

For ELISA-based detection:

• Human serum samples should be collected in serum separator tubes

• Allow samples to clot for 30 minutes before centrifugation

• Centrifuge at 1000 g for 15 minutes

• Aliquot and store at -80°C to avoid freeze-thaw cycles

• Dilute samples appropriately within the kit's detection range (78.13-5000 pg/mL)

For all methods:

• Avoid repeated freeze-thaw cycles

• Process all samples consistently to minimize technical variation

• Include appropriate extraction controls for each batch

Following these methodological guidelines will ensure sample integrity and reliable FERMT1 detection results.

How does FERMT1 contribute to EMT and what antibody applications can effectively study this process?

FERMT1 plays a significant role in epithelial-mesenchymal transition (EMT), a process crucial for cancer invasion and metastasis. Research indicates that FERMT1 mediates EMT through multiple mechanisms:

FERMT1's role in EMT:

• Downregulation of epithelial markers (E-cadherin)

• Upregulation of mesenchymal markers (N-cadherin, vimentin)

• Direct binding to NLRP3 and inhibition of NF-κB signaling pathway

• In colon cancer, activation of β-catenin transcriptional activity

• In NSCLC, upregulation of plakophilin 3 (PKP3) and activation of p38 MAPK signaling

Antibody applications to study FERMT1-mediated EMT:

• Multi-parameter IHC/IF: Co-staining FERMT1 with EMT markers (E-cadherin, N-cadherin, vimentin) to visualize correlations within the same tissue section. Use antibody combinations like anti-FERMT1 (22215-1-AP, Proteintech) with anti-E-cadherin (14472, Cell Signaling Technology) .

• Co-immunoprecipitation (Co-IP): For studying FERMT1 protein interactions with EMT regulators like NLRP3. This requires antibodies suitable for immunoprecipitation that won't interfere with protein-protein interaction domains .

• ChIP assays: To investigate whether FERMT1 associates with transcriptional complexes that regulate EMT genes, utilizing ChIP-grade FERMT1 antibodies.

• Proximity ligation assay (PLA): To visualize and quantify in situ FERMT1 interactions with EMT regulators at the single-molecule level.

When designing experiments, include proper controls and validate antibody specificity for each application to ensure reliable interpretation of FERMT1's role in EMT.

What approaches should be used to resolve contradictory FERMT1 expression data between different detection methods?

Contradictory FERMT1 expression data between different detection methods is a common research challenge. A systematic troubleshooting approach includes:

Methodological reconciliation strategy:

• Antibody validation assessment:

  • Verify antibody specificity using FERMT1 knockdown or knockout controls

  • Test multiple antibody clones targeting different epitopes

  • Review antibody validation data from manufacturers

  • Perform peptide blocking experiments to confirm specificity

• Technical considerations by method:

  • Western blot vs. IHC discrepancies:

    • Western blot measures total protein from homogenized samples

    • IHC reveals spatial distribution and heterogeneity within tissues

    • Consider cell-type specific expression that might be diluted in whole-tissue lysates

  • RT-PCR vs. protein detection discrepancies:

    • Assess post-transcriptional regulation mechanisms

    • Examine protein stability and turnover rates

    • Investigate miRNA regulation of FERMT1 mRNA

• Experimental design improvements:

  • Use matched samples for all techniques

  • Perform biological replicates (n≥3) and technical replicates

  • Include appropriate positive and negative controls

  • Quantify results using standardized scoring systems or densitometry

• Advanced resolution approaches:

  • Single-cell analysis techniques to address heterogeneity

  • Laser capture microdissection to isolate specific cell populations

  • Orthogonal validation using genetic manipulation (siRNA, CRISPR)

• Reporting recommendations:

  • Transparently report contradictory findings

  • Provide detailed methodological information

  • Discuss biological implications of the discrepancies

This systematic approach will help researchers reconcile contradictory data and strengthen the validity of FERMT1 expression findings.

How can FERMT1 antibodies be utilized to investigate its interaction with NLRP3 inflammasome components?

The interaction between FERMT1 and NLRP3 represents an important mechanism in cancer pathogenesis. Designing experiments to investigate this interaction requires careful antibody selection and experimental design:

Co-immunoprecipitation (Co-IP) methodology:

• Use antibodies specifically validated for immunoprecipitation

• Perform reciprocal Co-IPs (FERMT1 pull-down → NLRP3 detection and vice versa)

• Include proper negative controls (IgG, irrelevant antibody)

• Use mild lysis conditions to preserve protein-protein interactions

• Consider crosslinking to stabilize transient interactions

Research has demonstrated that FERMT1 directly binds to NLRP3, inhibiting the NF-κB signaling pathway . This interaction affects downstream processes including EMT and cancer progression.

Control experiments:

• Input controls (5-10% of lysate used for IP)

• IgG controls to assess non-specific binding

• Knockdown/knockout controls to validate specificity

• Competition assays with recombinant proteins

Advanced interaction mapping:

• Domain mapping using truncated constructs

• Mutagenesis of key residues to identify interaction sites

• Proximity ligation assay for in situ visualization of the interaction

• FRET/BRET assays to study the interaction dynamics in living cells

Data analysis considerations:

• Quantify Co-IP efficiency under different experimental conditions

• Assess the effect of stimuli that activate NLRP3 inflammasome

• Investigate how the interaction changes in different cancer contexts

This methodological framework provides a robust approach to characterize the FERMT1-NLRP3 interaction, which may represent a novel therapeutic target in cancer treatment.

What are the optimal experimental designs for studying FERMT1's role in cancer cell invasion and migration?

Studying FERMT1's role in cancer cell invasion and migration requires carefully designed experiments that can quantitatively assess these phenotypes while manipulating FERMT1 expression:

Experimental setup:

• FERMT1 expression modulation:

  • Knockdown using validated siRNAs or shRNAs targeting FERMT1

  • Overexpression using appropriate expression vectors (e.g., pcDNA3)

  • CRISPR/Cas9-mediated knockout for complete ablation

  • Rescue experiments to confirm specificity

• Migration assays:

  • Wound healing assay: Simple approach to assess collective migration

    • Create standardized "wounds" using scratch or inserts

    • Image at regular intervals (0, 24, 48, 72 hours)

    • Quantify wound closure rate using image analysis software

  • Transwell migration assay: For directional cell migration

    • Use appropriate pore size (typically 8 μm)

    • Optimize seeding density and migration time

    • Quantify migrated cells after staining

• Invasion assays:

  • Matrigel-coated transwell assay: Standard approach

    • Use growth factor-reduced Matrigel at optimized concentration

    • Include migration control (non-coated transwell)

    • Calculate invasion index (ratio of invaded to migrated cells)

  • 3D spheroid invasion assay: More physiologically relevant

    • Form tumor spheroids in low-attachment plates

    • Embed in matrix (Matrigel, collagen, or mixture)

    • Measure invasion distance over time

• Controls and validation:

  • Include positive controls (known pro-invasive factors)

  • Validate FERMT1 modulation by Western blot

  • Assess cell viability to exclude proliferation effects

  • Test in multiple cell lines to establish generalizability

Research has demonstrated that FERMT1 knockdown significantly decreases migration and invasion of nasopharyngeal carcinoma cells and non-small cell lung cancer cells . Conversely, FERMT1-overexpressing cells exhibit enhanced invasive ability compared to FERMT2- and FERMT3-overexpressing cells .

This comprehensive experimental approach will yield robust insights into FERMT1's role in cancer cell invasion and migration.

How can gene set enrichment analysis be applied to interpret FERMT1-associated pathways in cancer?

Gene Set Enrichment Analysis (GSEA) is a powerful computational method to identify significantly enriched biological pathways associated with FERMT1 expression in cancer. Based on published methodologies, researchers should:

GSEA implementation protocol:

• Dataset preparation:

  • Generate gene expression data from FERMT1-modulated cells

  • Alternatively, use public databases (TCGA, GEO) and stratify samples by FERMT1 expression

  • Ensure proper normalization and quality control of expression data

• GSEA execution:

  • Use GSEA software from UC San Diego and Broad Institute

  • Apply gene set permutations (1000 times per analysis)

  • Focus on hallmark gene sets for initial analysis

  • Consider pathway-specific gene sets based on research focus

• Result interpretation:

  • Evaluate normalized enrichment score (NES)

  • Assess statistical significance (nominal P-value)

  • Control for multiple testing using false discovery rate (FDR) q-value

  • Visualize enrichment plots for significant pathways

• Validation approaches:

  • Experimental validation of key pathway members

  • Protein-level confirmation of pathway activation

  • Pharmacological inhibition of identified pathways

  • Correlation with clinical outcomes

Published research has applied GSEA to identify FERMT1-associated pathways in nasopharyngeal carcinoma, revealing connections to EMT and cell cycle regulation . In non-small cell lung cancer, FERMT1 was linked to p38 MAPK pathway activation .

Practical considerations:

• Distinguish between pathways directly regulated by FERMT1 versus indirect associations

• Examine tissue-specific pathway enrichment patterns

• Consider the impact of tumor microenvironment on pathway activation

• Integrate with other omics data (proteomics, epigenomics) for comprehensive understanding

This methodological approach provides a framework for discovering and validating FERMT1-associated pathways, potentially revealing new therapeutic targets in cancer.

What quality control measures should be implemented when developing FERMT1 antibody-based diagnostic assays?

Developing reliable FERMT1 antibody-based diagnostic assays requires rigorous quality control measures to ensure reproducibility, specificity, and clinical utility:

Antibody validation requirements:

• Validate antibody specificity using multiple methods (Western blot, IHC, ELISA)

• Test against FERMT1-knockout or knockdown controls

• Assess cross-reactivity with other fermitin family members (FERMT2, FERMT3)

• Evaluate batch-to-batch consistency using reference standards

Assay development quality metrics:

• For ELISA-based assays:

  • Sensitivity: Achieve detection limits below 39 pg/mL

  • Detection range: Establish working range (e.g., 78.13-5000 pg/mL)

  • Precision:

    • Intra-plate: CV<10%

    • Inter-plate: CV<15%

  • Recovery: 80-120% across various matrices

  • Specificity: No significant cross-reactivity with analogues

• For IHC-based assays:

  • Establish clear scoring criteria (0 to 3+ system)

  • Ensure inter-observer concordance (>85%)

  • Include external quality assessment

  • Use tissue microarrays for standardization

Clinical validation parameters:

• Determine reference ranges in healthy populations

• Assess clinical sensitivity and specificity

• Calculate positive and negative predictive values

• Evaluate reproducibility across clinical laboratories

Implementation considerations:

• Standard operating procedures for pre-analytical variables

• Regular calibration and maintenance protocols

• Proficiency testing and external quality assessment

• Ongoing monitoring of assay performance

These quality control measures will ensure that FERMT1 antibody-based diagnostic assays provide reliable data for clinical decision-making and research applications.

How can FERMT1 antibodies be used to stratify cancer patients for potential targeted therapies?

FERMT1 expression correlates with poor prognosis in multiple cancer types, making it a potential biomarker for patient stratification. Developing effective stratification protocols using FERMT1 antibodies requires:

Patient stratification methodology:

Potential therapeutic implications:

• Patients with high FERMT1 expression might benefit from therapies targeting EMT

• FERMT1 status could predict response to p38 MAPK inhibitors

• Combined assessment of FERMT1 with NLRP3 expression might inform inflammasome-targeted therapies

This approach provides a framework for translating FERMT1 research into clinically relevant patient stratification strategies.

What are the technical considerations for developing phospho-specific FERMT1 antibodies?

Developing phospho-specific FERMT1 antibodies represents an advanced research tool to study activation states of FERMT1. The process requires:

Development strategy:

• Phosphorylation site identification:

  • Analyze known FERMT1 phosphorylation sites from phosphoproteomic studies

  • Conduct in silico analysis to identify conserved kinase motifs

  • Perform mass spectrometry to identify novel phosphorylation sites

  • Prioritize sites based on:

    • Conservation across species

    • Structural significance

    • Regulatory potential

• Phospho-peptide design:

  • Generate 10-15 amino acid peptides containing the phosphorylated residue

  • Include carrier protein for enhanced immunogenicity

  • Consider both phosphorylated and non-phosphorylated peptides for screening

• Antibody production and screening:

  • Immunize multiple rabbits to generate polyclonal antibodies

  • Screen using:

    • Phosphopeptide ELISA (phospho vs. non-phospho peptides)

    • Western blot of phosphatase-treated vs. untreated lysates

    • Immunoprecipitation followed by phosphatase treatment

• Validation in biological contexts:

  • Test in cells treated with pathway activators/inhibitors

  • Verify specificity in FERMT1 knockout backgrounds

  • Validate using phospho-mimetic and phospho-dead mutants

Application considerations:

• Establish optimal conditions for sample preparation to preserve phosphorylation

• Include phosphatase inhibitors in all buffers

• Consider using phos-tag gels for enhanced separation of phosphorylated species

• Develop quantitative assays to measure phosphorylation dynamics

This methodological approach will enable researchers to study FERMT1 activation in various cancer contexts and potentially identify new therapeutic strategies targeting specific activation states.

How can FERMT1 expression be effectively quantified in heterogeneous tumor samples?

Heterogeneous tumor samples present challenges for accurate FERMT1 quantification. Advanced methodological approaches include:

Quantification strategies for heterogeneous samples:

• Digital spatial profiling:

  • Combine FERMT1 antibody with spatial transcriptomics

  • Map FERMT1 expression across different tumor regions

  • Correlate with histopathological features

  • Integrate with other biomarkers for comprehensive profiling

• Single-cell analysis:

  • Dissociate tumor samples into single-cell suspensions

  • Perform flow cytometry using validated FERMT1 antibodies

  • Combine with lineage markers to identify cell-specific expression

  • Consider single-cell Western blotting for protein-level analysis

• Advanced image analysis for IHC:

  • Use whole slide imaging and automated analysis

  • Implement machine learning algorithms for pattern recognition

  • Quantify FERMT1 expression in specific tumor compartments

  • Consider multiplex IHC to correlate with other markers

• Laser capture microdissection:

  • Isolate specific regions of interest from tumor sections

  • Extract proteins or RNA for FERMT1 quantification

  • Compare expression between tumor center, invasive front, and stroma

  • Correlate with histopathological and clinical features

Data integration approaches:

• Weighted scoring systems that account for tumor heterogeneity

• Multi-parameter analysis combining FERMT1 with EMT markers

• Correlation with genetic and epigenetic profiles

• Integration with clinical outcome data

This comprehensive approach addresses the challenges of tumor heterogeneity, providing more accurate and clinically relevant FERMT1 quantification for research and potential diagnostic applications.