FREM2 Antibody, HRP conjugated

What is FREM2 and why is it significant in research?

FREM2 (FRAS1-related extracellular matrix protein 2) is an essential extracellular matrix protein required for maintaining the integrity of skin and renal epithelia. It plays a critical role in epidermal adhesion and is involved in the development of eyelids and the anterior segment of eyeballs. Its significance extends to multiple research areas including developmental biology, epithelial biology, and cancer research. FREM2 functions within a ternary complex alongside FRAS1 and FREM1, which is required for normal embryogenesis. Understanding FREM2 is particularly valuable for researchers studying epithelial integrity, congenital anomalies, and specific cancer types where FREM2 mutations have been identified .

What applications are FREM2 antibodies most commonly used for?

FREM2 antibodies are predominantly utilized in immunohistochemistry on paraffin-embedded tissues (IHC-P), where they effectively visualize FREM2 expression patterns in human tissues. The most validated applications include detection of FREM2 in breast and kidney tissues, with established protocols using 5 μg/ml concentration for optimal staining. While immunohistochemistry represents the primary validated application, researchers have also employed FREM2 antibodies in Western blotting (with dilutions around 1:500) and immunofluorescence microscopy (typically at 1:30 dilution). These applications have been instrumental in examining FREM2 expression in contexts ranging from developmental studies to cancer tissue analysis .

How should researchers prepare samples for optimal FREM2 antibody staining?

For optimal FREM2 antibody staining in immunohistochemistry, tissues should be fixed in 4% formaldehyde (or 10% neutral buffered formalin) for 15-24 hours before paraffin embedding. Tissue sections of 4-5 μm thickness provide optimal results. Antigen retrieval is typically performed using heat-induced epitope retrieval methods with citrate buffer (pH 6.0). For immunofluorescence applications with cultured cells, fixation in 4% formaldehyde for 15 minutes at room temperature followed by permeabilization with 0.1% Triton X-100 for 15 minutes and blocking with 1% BSA has been successfully employed. This preparation protocol has been validated in multiple studies examining FREM2 expression in various tissue contexts and cell lines .

What controls should be included when using FREM2 antibodies?

When working with FREM2 antibodies, researchers should implement a comprehensive control strategy including: (1) Positive tissue controls such as human breast or kidney tissues where FREM2 expression has been well-documented; (2) Negative controls by omitting the primary antibody while maintaining all other steps; (3) Isotype controls using non-specific IgG of the same species and at the same concentration as the FREM2 antibody; and (4) Antibody validation controls when possible, such as tissues from FREM2 knockout models or cells with FREM2 knockdown. For HRP-conjugated antibodies specifically, additional controls for endogenous peroxidase activity should be included by pre-treating sections with hydrogen peroxide. These controls are essential for distinguishing specific staining from background and ensuring experimental reliability .

How do mutations in FREM2 affect antibody binding and experimental interpretation?

Mutations in FREM2 can significantly impact antibody binding efficiency and experimental interpretation, particularly with antibodies targeting specific epitopes. For example, the p.Arg2167Trp missense mutation alters a conserved residue in FREM2, potentially changing the protein's conformation and accessibility of epitopes. Similarly, the p.Glu1972Lys mutation associated with Fraser syndrome affects FREM2-FREM1 interactions. When working with tissues harboring such mutations, researchers should consider using antibodies targeting multiple epitopes across the FREM2 protein to ensure detection. Additionally, quantitative analysis should account for potential alterations in antibody affinity. For research on mutation-specific effects, paired comparative studies using both wild-type and mutant-specific antibodies may provide more comprehensive insights into the functional consequences of these mutations .

What methodological modifications are needed for detecting FREM2 in different cancer types?

Cancer tissues often present unique challenges for FREM2 detection due to altered protein expression, post-translational modifications, and complex tumor microenvironments. For colorectal cancer samples, where FREM2 mutations have been associated with worse prognosis, researchers should consider extended antigen retrieval (20 minutes vs. standard 15 minutes) and optimize antibody concentration (3-7 μg/ml range). For glioblastoma tissues, where FREM2 shows higher expression in stem-like cell lines, membrane permeabilization protocols may need adjustment, and dual staining with stem cell markers can provide valuable context. Additionally, multiplexed immunofluorescence approaches combining FREM2 antibodies with markers of tumor microenvironment can reveal important interactions. In all cancer applications, careful validation against RNA expression data is recommended to confirm specificity of detection within the altered cellular context .

How should researchers troubleshoot inconsistent FREM2 staining patterns in experimental tissues?

When encountering inconsistent FREM2 staining patterns, researchers should implement a systematic troubleshooting approach addressing: (1) Tissue fixation duration—overfixation (>24 hours) can mask epitopes while underfixation (<12 hours) can compromise tissue morphology; (2) Antigen retrieval parameters—optimize pH (6.0 vs. 9.0) and duration (10-30 minutes) through parallel testing; (3) Antibody concentration—perform titration experiments across 1-10 μg/ml range; (4) Incubation conditions—evaluate temperature effects (4°C overnight vs. room temperature for 1-2 hours); and (5) Detection system sensitivity—compare amplification systems for low-expressing samples. For HRP-conjugated antibodies specifically, researchers should evaluate potential quenching of HRP activity in tissues with high endogenous peroxidase activity and consider alternative blocking reagents. Sample-specific optimization is particularly important when comparing tissues with varying FREM2 expression levels such as embryonic versus adult tissues .

How does HRP conjugation affect FREM2 antibody sensitivity and specificity?

HRP conjugation can influence both sensitivity and specificity parameters of FREM2 antibodies through several mechanisms. Regarding sensitivity, direct HRP conjugation may reduce detection sensitivity by 10-30% compared to amplified secondary systems due to fewer HRP molecules per antibody binding event. This is particularly relevant when detecting FREM2 in tissues with naturally low expression levels. For specificity considerations, while HRP conjugation eliminates potential cross-reactivity from secondary antibodies, the conjugation process itself can occasionally affect the antibody's paratope, subtly altering epitope recognition. Researchers should validate HRP-conjugated FREM2 antibodies against established unconjugated versions using parallel tissue sections to establish comparative detection thresholds. To maximize both parameters, optimal dilution testing across a broader range (1:20 to 1:500) is recommended, with particular attention to background levels in negative control tissues .

What substrate systems work optimally with HRP-conjugated FREM2 antibodies?

For HRP-conjugated FREM2 antibodies, substrate selection significantly impacts detection sensitivity and signal persistence. Diaminobenzidine (DAB) remains the standard substrate for most applications, providing a stable brown precipitate with good contrast against hematoxylin counterstaining in epithelial tissues where FREM2 is commonly studied. For enhanced sensitivity in tissues with lower FREM2 expression, amplified DAB systems incorporating nickel or copper enhancement can increase detection threshold by 2-3 fold. When studying FREM2 in tissues with high melanin content (which can obscure DAB signals), alternative chromogens such as AEC (red) or Vector VIP (purple) provide better distinction. For quantitative applications, researchers should consider enhanced chemiluminescent (ECL) substrates, which offer superior dynamic range for densitometric analysis. Substrate incubation times should be carefully optimized, with typical ranges of 3-5 minutes for standard DAB and 30-60 seconds for high-sensitivity substrates to prevent overreaction and non-specific background .

How can researchers quantitatively analyze FREM2 expression patterns in tissue samples?

Quantitative analysis of FREM2 expression requires standardized approaches to ensure reliability across experiments. A multi-parameter assessment framework is recommended, incorporating: (1) Staining intensity scoring on a 0-3 scale (0=negative, 1=weak, 2=moderate, 3=strong); (2) Percentage of positive cells within the tissue section; (3) Calculation of H-scores (intensity × percentage, ranging from 0-300); and (4) Subcellular localization patterns (membrane, cytoplasmic, or mixed). Digital image analysis offers more objective assessment, with color deconvolution algorithms separating DAB signal from counterstains for pixel-based quantification. For correlation with clinical parameters, researchers should normalize FREM2 expression against appropriate housekeeping proteins. When comparing different tissue types, it's essential to account for variations in background staining by implementing tissue-specific thresholding. This comprehensive approach has successfully been applied in studies examining FREM2 expression across normal tissues, developmental stages, and pathological conditions .

What unique challenges exist when studying FREM2 in different developmental contexts?

Studying FREM2 across developmental contexts presents distinct methodological challenges requiring specialized approaches. During embryonic development, FREM2's expression in neural stem cells and various epithelia necessitates careful stage-specific analysis. Researchers should employ microdissection techniques to isolate relevant tissue regions before antibody application, as FREM2's spatial expression pattern changes significantly across developmental timepoints. Embryonic tissues typically require shorter fixation times (6-12 hours) compared to adult tissues to preserve antigenicity while maintaining structural integrity. For detecting the FREM2-FREM1-FRAS1 complex formation during development, proximity ligation assays provide valuable insights beyond standard co-localization studies. When comparing expression between embryonic and adult tissues, standardized quantification becomes crucial, as baseline expression levels differ substantially. These developmental studies have been instrumental in understanding FREM2's role in congenital conditions such as cryptophthalmos and renal agenesis, highlighting the importance of methodological adaptations for developmental research .

How should researchers interpret discrepancies between FREM2 protein detection and gene expression data?

When confronting discrepancies between FREM2 protein detection using antibodies and corresponding gene expression data, researchers should systematically evaluate several potential mechanisms. Post-transcriptional regulation through microRNAs targeting FREM2 mRNA can result in reduced protein despite abundant transcript. Similarly, post-translational modifications may affect epitope accessibility, particularly in the large FREM2 protein with numerous glycosylation sites. Research has shown that mutations such as c.15delG can trigger nonsense-mediated mRNA decay, resulting in transcript detection but minimal protein. Tissue-specific protein turnover rates may also contribute to observed differences. To reconcile such discrepancies, researchers should implement a multi-method validation approach combining: (1) Antibodies targeting different FREM2 epitopes; (2) Western blotting to confirm protein size; (3) RNA-seq or qRT-PCR with exon-spanning primers; and (4) Polysome profiling to assess translation efficiency. This integrated approach has successfully resolved apparent contradictions in FREM2 expression patterns across different experimental contexts .

What are the optimal experimental conditions for detecting FREM2 in co-immunoprecipitation studies?

For successful co-immunoprecipitation (Co-IP) studies investigating FREM2 interactions, particularly with FREM1 and FRAS1 proteins, researchers should implement specific optimization strategies. Lysis buffer composition is critical—a modified RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) supplemented with protease inhibitors prevents complex dissociation while efficiently extracting membrane-associated proteins. Pre-clearing lysates with protein A/G beads for 1 hour significantly reduces non-specific binding. For antibody selection, using approximately 5 μg of FREM2 antibody per 500 μg of total protein yields optimal results. Extended incubation (overnight at 4°C with gentle rotation) is essential for capturing the large FREM2 protein complexes. Washing conditions should be stringent enough to remove non-specific interactions but gentle enough to preserve authentic complexes—typically four washes with decreasing salt concentrations (from 300 mM to 150 mM NaCl). These optimized conditions have successfully demonstrated the functional significance of FREM2-FREM1 interactions and how mutations such as p.Arg2167Trp can impair these protein-protein associations .

How can researchers effectively distinguish between FREM2 and other FREM family proteins in experimental systems?

Distinguishing between FREM2 and other FREM family proteins (particularly FREM1 and FREM3) requires careful consideration of antibody specificity and experimental controls. Sequence alignment analysis reveals several regions of homology between FREM family proteins, creating potential cross-reactivity. Researchers should select antibodies targeting the least conserved regions, particularly within the unique N-terminal domains of FREM2. Validation through parallel experiments with FREM1, FREM2, and FREM3 knockout or knockdown systems provides definitive specificity confirmation. For immunohistochemistry applications, sequential tissue sections stained with antibodies against different FREM proteins help establish distinct distribution patterns. In Western blotting, FREM2 (~350 kDa) can be distinguished from FREM1 (~314 kDa) and FREM3 (~211 kDa) by molecular weight. For RNA-based detection methods, primer design should target unique exons with minimal sequence similarity. This multi-faceted approach ensures accurate discrimination between FREM family members, which is essential given their different but sometimes overlapping functions in developmental processes and disease contexts .

What methodological adaptations are necessary when using FREM2 antibodies in different species?

Cross-species applications of FREM2 antibodies require methodological adaptations to account for sequence variations and tissue-specific differences. While human and mouse FREM2 share approximately 88% sequence identity, critical epitope regions may differ. For rodent tissues, antibody concentration typically needs to be increased by 25-50% compared to human samples (approximately 6-8 μg/ml versus 5 μg/ml for IHC-P). Antigen retrieval conditions often require optimization, with mouse tissues generally benefiting from slightly longer retrieval times (20 minutes versus 15 minutes) and higher pH buffer systems (pH 9.0 versus pH 6.0). Detection systems may need enhancement through amplification steps such as biotinylated tyramide signal amplification when working with species having lower sequence homology to the immunogen. For non-mammalian species, researchers should first perform in silico epitope mapping to predict potential cross-reactivity, followed by extensive validation including Western blotting to confirm specific binding. Importantly, interpretation of staining patterns must consider species-specific expression domains, as FREM2 distribution varies particularly during developmental stages across different organisms .

How can FREM2 antibodies be incorporated into multiplexed immunofluorescence systems?

Incorporating FREM2 antibodies into multiplexed immunofluorescence systems requires strategic protocol adaptations to maintain signal specificity while enabling visualization of multiple targets. For effective multiplexing, researchers should consider: (1) Primary antibody species selection—pairing rabbit-derived FREM2 antibodies with mouse or goat antibodies against other targets minimizes cross-reactivity; (2) Sequential antibody application with microwave treatment (3 minutes at 800W in citrate buffer) between rounds to strip previous antibodies while preserving tissue architecture; (3) Careful fluorophore selection—pairing HRP-conjugated FREM2 antibodies with tyramide signal amplification systems using spectrally distinct fluorophores (such as Cy3 for FREM2 and Alexa Fluor 488 for other targets); and (4) Implementation of spectral unmixing algorithms during image acquisition to resolve overlapping emission spectra. This approach has been successfully employed to simultaneously visualize FREM2 alongside proliferation markers (Ki-67), cell-type specific markers, and other extracellular matrix components, providing valuable insights into FREM2's contextual relationships within complex tissue microenvironments .

What approaches can be used to study the functional impact of FREM2 mutations on protein-protein interactions?

Studying the functional consequences of FREM2 mutations on protein-protein interactions requires sophisticated methodological approaches beyond standard detection. Researchers have successfully employed: (1) FRET (Förster Resonance Energy Transfer) assays using fluorophore-tagged FREM2 (wild-type and mutant variants) and potential binding partners to measure interaction distances with nanometer precision; (2) Surface Plasmon Resonance (SPR) to quantify binding kinetics between purified FREM2 variants and partners like FREM1, revealing how mutations such as p.Arg2167Trp reduce association rates; (3) Mammalian two-hybrid systems for high-throughput screening of interaction networks affected by specific mutations; and (4) Proximity ligation assays in fixed tissues or cells to visualize and quantify protein complexes in situ. These methods have demonstrated that mutations like p.Arg2167Trp impair FREM2-FREM1 interactions less severely than the Fraser syndrome-associated p.Glu1972Lys mutation, providing molecular explanations for phenotypic differences between isolated cryptophthalmos and more severe systemic conditions. Such functional assessments are essential for translating genetic findings into mechanistic understanding of disease pathogenesis .

How can researchers design effective FREM2 knockdown validation experiments using antibody detection methods?

Designing effective FREM2 knockdown validation experiments requires comprehensive controls and quantification methods. A robust validation framework should include: (1) Multiple knockdown approaches—comparing siRNA, shRNA, and CRISPR-Cas9 methods to control for off-target effects; (2) Dose-response assessment—testing different concentrations of knockdown reagents to establish correlation between treatment intensity and FREM2 reduction; (3) Time-course analysis—measuring FREM2 levels at 24, 48, 72, and 96 hours post-treatment to determine optimal assessment timepoints; and (4) Parallel quantification via Western blotting (for total protein levels) and immunofluorescence (for localization changes). Critically, researchers should employ at least two different FREM2 antibodies targeting distinct epitopes to confirm consistent knockdown patterns. Additionally, rescue experiments reintroducing wild-type FREM2 provide definitive validation of phenotypic specificity. For HRP-conjugated antibodies, chemiluminescent substrates offer superior quantitative assessment of knockdown efficiency compared to chromogenic detection methods. This systematic approach ensures reliable validation of FREM2 knockdown, which is essential for subsequent functional studies in cancer research, developmental biology, and epithelial biology contexts .

Comparative Analysis Table: Applications of Different FREM2 Antibody Types

This table provides researchers with a comparative framework for selecting the appropriate FREM2 antibody type based on specific experimental requirements and tissue characteristics .

How might emerging antibody technologies enhance FREM2 detection and functional analysis?

Emerging antibody technologies offer promising opportunities to advance FREM2 research beyond current limitations. Nanobody-based detection systems, with their smaller size (~15 kDa versus ~150 kDa for conventional antibodies), provide superior tissue penetration for detecting FREM2 in intact three-dimensional tissue models and organoids. These smaller binding molecules can access epitopes that might be sterically hindered in the large FREM2 protein complex. BiTE (Bi-specific T-cell Engager) antibody constructs combining FREM2-targeting domains with domains recognizing specific cellular markers could enable precise localization studies. Additionally, photoswitchable antibody systems would allow time-resolved tracking of FREM2 trafficking and turnover in live cells. For quantitative applications, emerging mass cytometry (CyTOF) compatible antibodies tagged with rare earth metals rather than fluorophores or enzymes would enable highly multiplexed analysis of FREM2 alongside dozens of other proteins without spectral overlap limitations. These technologies will be particularly valuable for studying FREM2's dynamic interactions during development and in disease processes, potentially revealing previously unrecognized functions of this important extracellular matrix protein .

What are the potential applications of FREM2 antibodies in cancer biomarker research?

FREM2 antibodies hold significant potential for cancer biomarker applications based on emerging evidence of FREM2's role in multiple cancer types. In colorectal cancer, where FREM2 mutations correlate with poorer prognosis, standardized IHC protocols using HRP-conjugated FREM2 antibodies could enable stratification of patients for targeted therapies. For glioblastoma, where FREM2 expression is elevated in stem-like cell populations, FREM2 antibodies could help identify tumor-initiating cell populations with therapeutic resistance. Methodologically, researchers should focus on developing tissue microarray-compatible protocols with digital pathology quantification to establish clinically relevant expression thresholds. Combining FREM2 detection with established cancer biomarkers in multiplexed panels would provide contextual information on tumor microenvironment interactions. Additionally, development of circulating tumor cell detection methods incorporating FREM2 antibodies could enable liquid biopsy approaches for minimally invasive monitoring. As research progresses, validation of these applications will require multi-institutional studies with standardized antibody protocols to establish reproducible clinicopathological correlations across diverse patient populations .