FREM2 Antibody, Biotin conjugated

What is FREM2 and what are its primary biological functions?

FREM2 is an extracellular matrix protein that is crucial for maintaining the integrity of skin epithelium and renal epithelia. It plays an essential role in epidermal adhesion, forming part of the basement membrane complex . Research has demonstrated that FREM2 is significantly involved in the development of eyelids and the anterior segment of eyeballs . At the molecular level, FREM2 contains numerous CSPG (chondroitin sulfate proteoglycan element) repeats and Calx-beta domains that contribute to its structural and functional properties .

The protein localizes to the basement membrane, forming a ternary complex that mediates epidermal-dermal interactions . Recent studies have also established that FREM2, as part of the Fraser complex proteins, forms anchoring cords that temporally stabilize the dermal-epidermal junction during embryonic development . These functions position FREM2 as a critical component in tissue integrity and development pathways.

How does a biotin-conjugated antibody differ from unconjugated FREM2 antibodies?

Biotin-conjugated FREM2 antibodies have biotin molecules covalently attached to the antibody structure, enabling detection via biotin-binding proteins such as streptavidin or avidin. The specific advantage of biotin-conjugated antibodies, particularly those with a spacer arm (Biotin-SP) like the FREM2 Antibody, Biotin conjugated, is the significantly enhanced sensitivity in detection systems .

The 6-atom spacer positioned between biotin and the antibody reduces steric hindrance, allowing more efficient binding of streptavidin to biotin. This structural modification results in improved signal amplification in enzyme immunoassays compared to antibodies conjugated with biotin without a spacer . For researchers investigating FREM2 in tissues with low expression levels, this increased sensitivity can be crucial for obtaining reliable results.

What experimental applications are best suited for FREM2 Antibody, Biotin conjugated?

The actual optimal dilution should be determined empirically for each experimental system, as factors such as antigen density, sample permeability, and detection system can influence results .

How should I design validation experiments for FREM2 Antibody, Biotin conjugated?

Validation of the FREM2 Antibody, Biotin conjugated should follow a systematic approach to ensure specificity and reliability:

• Positive control selection: Use tissues or cell lines known to express FREM2, such as skin epithelium, renal epithelia, or specific developmental tissues like embryonic eyelids . Human breast and kidney tissues have been successfully used for FREM2 antibody validation .

• Negative control implementation: Include both technical negative controls (omitting primary antibody) and biological negative controls (tissues/cells with minimal FREM2 expression).

• Specificity assessment: When possible, compare staining patterns with another validated FREM2 antibody targeting a different epitope.

• Epitope blocking experiment: Pre-incubate the antibody with the immunizing peptide (in this case, the synthetic peptide within Human FREM2 used for immunization) , which should abolish specific staining.

• Antibody titration: Test a range of dilutions to determine optimal signal-to-noise ratio, starting with manufacturer recommendations but adjusting based on your specific application.

• Cross-reactivity assessment: If working with non-human samples, evaluate potential cross-reactivity based on sequence homology and empirical testing .

What are the optimal storage and handling conditions for maintaining FREM2 Antibody, Biotin conjugated activity?

Proper storage and handling are critical for maintaining antibody functionality:

Short-term storage (up to 6 weeks):

• Store the rehydrated antibody at 2-8°C as an undiluted liquid

• Prepare working dilutions on the day of use for maximum sensitivity

Long-term storage (extended periods):

• Option 1: Aliquot and freeze at -70°C or below to avoid repeated freeze-thaw cycles

• Option 2: Add an equal volume of glycerol (ACS grade or better) to achieve a final concentration of 50%, then store at -20°C as a liquid

Handling precautions:

• Centrifuge the rehydrated product if not clear before use

• Avoid repeated freeze-thaw cycles as this can denature the antibody and reduce activity

• When diluting, use appropriate buffers that maintain protein stability

• Consider adding carrier proteins (BSA) to dilute antibody solutions to prevent adsorption to tubes

• Follow manufacturer's expiration guidelines, typically one year from date of rehydration

How can I optimize signal detection when using FREM2 Antibody, Biotin conjugated in immunohistochemistry?

Optimizing signal detection requires careful consideration of multiple parameters:

• Antigen retrieval optimization: Since FREM2 is a basement membrane protein, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) should be tested to determine which provides optimal antigen accessibility.

• Blocking protocol optimization: To reduce background, use a combination of serum (matching the species of the secondary detection reagent) and proteins (BSA or casein) in your blocking solution.

• Biotin-streptavidin system selection: Choose appropriate streptavidin-conjugated detection systems based on your imaging capabilities:

  • Streptavidin-HRP with DAB for brightfield microscopy

  • Streptavidin-fluorophore conjugates for fluorescence detection

  • Streptavidin-AP with appropriate chromogens for applications requiring higher sensitivity

• Signal amplification strategies: For tissues with low FREM2 expression, consider:

  • Tyramide signal amplification (TSA) combined with streptavidin-HRP

  • Multi-layer detection using biotinylated anti-streptavidin followed by additional streptavidin-conjugate

• Counterstain selection: Choose counterstains that complement your detection system without interfering with signal visualization:

  • Hematoxylin for DAB-based detection

  • DAPI or Hoechst for fluorescence-based detection

• Mounting medium considerations: Use anti-fade mounting media for fluorescence detection to preserve signal during imaging and storage.

What are common causes of high background when using FREM2 Antibody, Biotin conjugated, and how can they be addressed?

High background is a frequent challenge with biotin-conjugated antibodies, with several potential causes and solutions:

For particularly challenging samples with high endogenous biotin (like kidney, liver, or brain tissues), consider alternative detection methods such as directly conjugated antibodies or non-biotin detection systems .

How can I troubleshoot weak or absent signal when using FREM2 Antibody, Biotin conjugated?

Weak or absent signals may result from various experimental factors:

• Epitope masking or destruction: Optimize antigen retrieval methods by testing different buffers, pH conditions, and retrieval times.

• Insufficient primary antibody concentration: The recommended dilution for ELISA (1:20,000 - 1:400,000) may need adjustment for other applications. Try more concentrated solutions (starting with 1:100 - 1:500) for immunohistochemistry.

• Inadequate incubation time or temperature: Extend primary antibody incubation to overnight at 4°C to enhance binding, particularly for tissue sections.

• Antibody degradation: Verify antibody activity using known positive controls; consider obtaining new antibody if degradation is suspected.

• Detection system issues: Ensure your streptavidin-conjugate is active and appropriately diluted; expired or improperly stored detection reagents can lead to signal loss.

• Sample preparation problems: Excessive fixation can mask epitopes; sample storage conditions can affect antigen preservation.

• Low target expression: FREM2 expression is developmental stage-specific and tissue-specific. Verify expected expression in your sample type based on literature.

• Inappropriate blocking: Certain blocking reagents can interfere with antibody-epitope interactions; try alternative blocking solutions.

What strategies can resolve specificity concerns with FREM2 Antibody, Biotin conjugated?

Addressing specificity concerns requires multiple validation approaches:

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application to samples; specific signals should be abolished while non-specific signals will remain.

• Knockout/knockdown validation: Test the antibody on samples with FREM2 knockout or knockdown; specific signals should be reduced or eliminated. If FREM2 knockout models are unavailable, consider siRNA knockdown in cell culture systems.

• Multiple antibody validation: Compare staining patterns with another FREM2 antibody targeting a different epitope; concordant patterns suggest specificity.

• Western blot correlation: Perform Western blotting alongside immunohistochemistry to correlate band detection with histological staining patterns. FREM2 is expected at approximately 350 kDa.

• Cross-species validation: If the antibody is predicted to work in multiple species based on sequence homology, compare staining patterns across species.

• Literature comparison: Compare your observed staining patterns with published FREM2 localization data; FREM2 should localize primarily to basement membranes, particularly at the dermal-epidermal junction .

How can FREM2 Antibody, Biotin conjugated be applied in developmental biology research?

FREM2 has significant developmental importance, making its antibody valuable for developmental biology studies:

• Spatiotemporal expression mapping: Track FREM2 expression patterns during embryonic development, particularly focusing on:

  • Skin development and basement membrane formation

  • Kidney morphogenesis

  • Eye development, especially eyelid formation and anterior segment development

  • Dermal-epidermal junction formation

• Fraser syndrome model investigations: Since FREM2 mutations are associated with Fraser syndrome , the antibody can be used to examine pathological alterations in protein localization and expression in animal models of this condition.

• Co-localization studies: Combine FREM2 antibody with markers for other basement membrane components to understand the assembly sequence and structural organization of the basement membrane during development.

• Epithelial-mesenchymal interaction studies: Investigate FREM2's role in mediating epithelial-mesenchymal interactions during organogenesis, particularly in contexts where epithelial budding or invagination occurs .

• Tissue recombination experiments: Use the antibody to track FREM2 expression in tissue recombination experiments to understand its regulatory mechanisms during development.

What considerations are important when using FREM2 Antibody, Biotin conjugated in multiplex immunofluorescence studies?

Multiplex immunofluorescence with FREM2 Antibody, Biotin conjugated requires careful planning:

• Detection system compatibility: Select fluorophore-conjugated streptavidin with emission spectra that don't overlap with other fluorophores in your multiplex panel.

• Sequential staining considerations: If using multiple biotin-conjugated antibodies, complete each biotin-streptavidin staining sequence (including a blocking step) before proceeding to the next biotin-conjugated antibody.

• Antibody cross-reactivity assessment: Test each antibody individually before combining to ensure no unexpected cross-reactivity occurs.

• Concentration optimization: Biotin-conjugated antibodies may require different concentrations in multiplex settings versus single-staining applications.

• Order of application: Apply the FREM2 antibody in the optimal sequence; typically lower-abundance targets should be detected first.

• Controls for spectral overlap: Include single-stain controls to assess and correct for spectral bleed-through during analysis.

• Tissue autofluorescence management: Implement autofluorescence reduction protocols such as Sudan Black B treatment or commercial autofluorescence quenchers.

How can FREM2 Antibody, Biotin conjugated be used to investigate Fraser Complex protein interactions?

The Fraser Complex consists of interacting proteins that form anchoring cords during development . FREM2 Antibody, Biotin conjugated can help elucidate these interactions:

• Co-immunoprecipitation studies: Use the antibody to pull down FREM2 and identify associated proteins within the Fraser Complex. The biotin conjugation facilitates recovery using streptavidin beads.

• Proximity ligation assays (PLA): Combine FREM2 Antibody, Biotin conjugated with antibodies against other Fraser Complex proteins (FREM1, FRAS1) to visualize and quantify protein-protein interactions at the subcellular level.

• FRET/FLIM analysis: When paired with appropriate fluorophore-conjugated streptavidin and other labeled antibodies, FREM2 Antibody can be used in Förster Resonance Energy Transfer or Fluorescence-Lifetime Imaging Microscopy to study molecular-scale interactions.

• Super-resolution microscopy: Apply the antibody in techniques like STORM or PALM to examine nanoscale organization of FREM2 relative to other basement membrane components.

• In situ interaction studies: Use the antibody to examine how FREM2 localization changes in models where other Fraser Complex proteins are absent or mutated.

• Protein complex isolation: Employ the antibody in affinity purification schemes to isolate intact Fraser Complex for proteomic analysis.

What protocols should be considered when examining FREM2 expression in renal and skin disease models?

When investigating FREM2 in disease contexts, consider these specialized protocols:

• Tissue-specific fixation optimization:

  • For skin: Brief fixation (4-6 hours) in 4% PFA followed by careful paraffin embedding to preserve dermal-epidermal junction integrity

  • For kidney: Consider fixation with Bouin's solution for improved antigen preservation in glomerular structures

• Co-labeling strategy:

  • For skin: Combine with antibodies against other basement membrane components (collagen IV, laminin) and epithelial markers (keratins)

  • For kidney: Co-label with podocyte markers (nephrin, podocin) and glomerular basement membrane components

• Section orientation and thickness:

  • For skin: Prepare 5-7 μm vertical sections to properly visualize the dermal-epidermal junction

  • For kidney: Consider 3-5 μm sections for glomerular visualization or thicker sections (10-15 μm) for tubular structure examination

• Image analysis parameters:

  • Quantify FREM2 signal intensity along the basement membrane

  • Measure continuity/disruption of FREM2 staining

  • Assess co-localization coefficients with other basement membrane proteins

• Disease-specific considerations:

  • In Fraser syndrome models: Compare FREM2 localization and expression levels between affected and unaffected tissues

  • In renal disease models: Examine FREM2 alterations in relation to proteinuria and glomerular basement membrane disruption

  • In skin blistering disorders: Assess FREM2 expression at blister formation sites

How might FREM2 Antibody, Biotin conjugated contribute to understanding the relationship between FREM2 and signaling pathways?

FREM2 has connections to Integrin and ERK signaling pathways , and the biotin-conjugated antibody can help elucidate these relationships:

• Pathway activation studies: Use the antibody to monitor FREM2 expression and localization changes following modulation of Integrin or ERK pathway activity.

• Co-immunoprecipitation of signaling complexes: Utilize the antibody to isolate FREM2-associated protein complexes for analysis of associated signaling components.

• Receptor clustering analysis: Apply super-resolution microscopy with the antibody to examine FREM2 clustering in relation to integrin receptors during signaling events.

• Phosphoproteomic analysis: Combine FREM2 immunoprecipitation with phosphoproteomic analysis to identify phosphorylation events associated with FREM2-mediated signaling.

• CRISPR-based functional genomics: Use the antibody to validate FREM2 knockout or mutation effects on downstream signaling pathways in CRISPR-edited cell lines or animal models.

• Mechanotransduction studies: Investigate FREM2's role in translating mechanical forces at the basement membrane to intracellular signaling events using the antibody to track protein dynamics.

What are emerging applications for FREM2 Antibody, Biotin conjugated in cancer research?

While FREM2's role in cancer is still being elucidated, several research directions may benefit from this antibody:

• Tumor basement membrane integrity studies: Examine FREM2 expression and localization in tumor-associated basement membranes to understand structural alterations during invasion.

• Cancer stem cell niche investigation: Study FREM2's potential role in creating specialized extracellular matrix environments that support cancer stem cell maintenance.

• Epithelial-mesenchymal transition (EMT) research: Investigate FREM2 expression changes during EMT, a process critical for cancer metastasis.

• Biomarker exploration: Assess FREM2 as a potential diagnostic or prognostic biomarker in cancers with basement membrane involvement.

• Therapeutic target validation: If FREM2 emerges as a potential therapeutic target, the antibody can be used to validate target engagement and expression in preclinical models.

• Tumor microenvironment studies: Examine how FREM2 contributes to the organization of the tumor microenvironment, particularly in the context of tumor-stromal interactions.

How can computational approaches enhance interpretation of FREM2 Antibody, Biotin conjugated experimental data?

Modern computational methods can significantly enhance FREM2 antibody research:

• Image analysis automation: Develop algorithms to quantify FREM2 staining patterns in tissue samples, enabling high-throughput analysis across large sample sets.

• 3D reconstruction: Generate three-dimensional models of FREM2 distribution in tissues using z-stack confocal images to better understand spatial organization.

• Machine learning classification: Train neural networks to recognize normal versus abnormal FREM2 expression patterns in disease models.

• Molecular dynamics simulation: Combine antibody epitope information with protein structure predictions to model FREM2 interactions with other basement membrane components.

• Multi-omics data integration: Correlate FREM2 protein expression data with transcriptomic and genetic data to construct comprehensive models of FREM2 regulation and function.

• Systems biology modeling: Incorporate FREM2 into mathematical models of basement membrane assembly and tissue morphogenesis.

• Virtual tissue modeling: Develop in silico models of tissues incorporating FREM2 expression data to predict effects of alterations on tissue integrity and function.