FBLN5 Antibody, Biotin conjugated

What is FBLN5 and why is it important in research?

Fibulin-5 (FBLN5) is an extracellular matrix glycoprotein secreted by various cell types including vascular smooth muscle cells, fibroblasts, and endothelial cells. It contains an Arg-Gly-Asp (RGD) motif and calcium-binding EGF-like domains, which facilitate its interaction with integrins and promotion of endothelial cell adhesion . FBLN5 has gained significant research interest due to its involvement in various physiological processes, including vascular development and tissue remodeling, as well as its implication in pathological conditions such as age-related macular degeneration . Recent studies have also identified FBLN5 as a key paracrine factor secreted by human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) that controls the development of Schwann cells (SCs), making it relevant for research on peripheral nervous system myelination .

What are the biochemical characteristics of FBLN5 antibodies?

FBLN5 antibodies are immunoglobulins designed to recognize and bind specifically to Fibulin-5 protein. The commercial biotin-conjugated FBLN5 antibody typically has the following characteristics:

This information is essential for researchers to ensure proper experimental design and interpretation of results when using FBLN5 antibodies .

How should FBLN5 antibody be stored and handled for optimal performance?

For optimal performance and longevity, biotin-conjugated FBLN5 antibody should be stored as aliquots at -20°C . The antibody is typically supplied in a buffer of 0.01 M PBS, pH 7.4, with 0.03% Proclin-300 and 50% glycerol . Repeated freeze-thaw cycles should be avoided as they can lead to denaturation and loss of antibody activity . For unconjugated FBLN5 antibodies, storage at -20°C in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 is recommended, with stability up to one year after shipment . Aliquoting is unnecessary for -20°C storage of smaller volumes (20μl), which may contain 0.1% BSA for stabilization .

How does the RGD motif in FBLN5 affect antibody binding and functionality?

The RGD (Arg-Gly-Asp) motif in FBLN5 plays a critical role in its interaction with integrins, mediating cellular adhesion and signaling . When designing or selecting antibodies for FBLN5 research, consideration of the epitope location relative to this motif is crucial. Antibodies targeting regions near or within the RGD motif may interfere with FBLN5's biological functions, potentially affecting experimental outcomes when studying integrin-dependent processes. Conversely, such antibodies can be deliberately used to block FBLN5-integrin interactions in functional studies. Recent research has revealed that the RGD motif of FBLN5 is necessary for RAC1 activation through binding to Integrin, which modulates actin remodeling essential for Schwann cell myelination . Therefore, researchers should carefully evaluate whether their experimental design requires preservation or inhibition of this interaction when selecting appropriate antibodies.

What are the critical considerations for validating FBLN5 antibody specificity in experimental systems?

Validating antibody specificity is essential for reliable experimental results. For FBLN5 antibodies, validation should include:

• Western blot analysis: Verification of a single band at the expected molecular weight (65-70 kDa for FBLN5, which differs from the calculated 50 kDa due to post-translational modifications) .

• Positive and negative controls: Using tissues known to express FBLN5 (such as heart, kidney, lung, and colon tissues) versus tissues with low expression .

• Knockdown or knockout validation: Testing the antibody in systems where FBLN5 expression has been reduced or eliminated to confirm specificity.

• Cross-reactivity testing: Especially important for studies spanning multiple species, as antibody reactivity may vary between human, mouse, and rat samples .

• Epitope mapping: Understanding which region of FBLN5 the antibody recognizes, particularly in relation to functional domains like the RGD motif or EGF-like domains.

• Protocol optimization: For each application (WB, IHC, ELISA), optimal antibody concentrations should be determined through titration experiments, as recommended dilutions vary significantly (1:2000-1:10000 for WB, 1:50-1:500 for IHC) .

How can researchers accurately quantify FBLN5 expression levels using biotin-conjugated antibodies?

Accurate quantification of FBLN5 expression using biotin-conjugated antibodies requires careful methodological considerations:

• ELISA optimization: As the primary application for biotin-conjugated FBLN5 antibodies, ELISA protocols should be optimized for sensitivity and specificity. Standard curves using recombinant FBLN5 protein should be established for quantification.

• Signal amplification systems: Biotin-streptavidin systems provide signal amplification, but require careful calibration to ensure linearity of response across the relevant concentration range.

• Reference standards: Including well-characterized samples with known FBLN5 expression levels helps normalize results across experiments.

• Background reduction: Non-specific binding should be minimized through appropriate blocking steps and control reactions.

• Cross-validation: Results should be validated using alternative detection methods or antibodies recognizing different epitopes.

In experimental contexts, ELISA has been successfully used to quantify FBLN5 secretion from WJ-MSCs, revealing increased FBLN5 secretion (from 12.5 ± 0.99 pg/mL to 18.41 ± 0.26 pg/mL) when co-cultured with S16 Schwann cells .

What are the experimental considerations when studying FBLN5 interactions with other extracellular matrix components?

When investigating FBLN5 interactions with other extracellular matrix components, researchers should consider:

• Co-immunoprecipitation protocols: For studying protein-protein interactions, optimized co-IP protocols using biotin-conjugated FBLN5 antibodies coupled with streptavidin beads can capture FBLN5 and its interacting partners.

• Pull-down assays: Recombinant FBLN5-V5-His fusion proteins, purified using metal-chelate chromatography with Ni-NTA columns, can be used in pull-down experiments to identify interaction partners .

• Surface plasmon resonance: For quantitative analysis of binding kinetics between FBLN5 and potential interactors.

• Native conditions preservation: Experimental conditions should preserve the native conformation of FBLN5, particularly the calcium-binding EGF-like domains that may be sensitive to calcium chelation.

• Competitive binding assays: Using synthetic peptides corresponding to the RGD motif to compete with FBLN5-integrin interactions.

• Cross-linking approaches: Chemical cross-linking followed by mass spectrometry can identify transient or weak interactions within the extracellular matrix environment.

Recent research has demonstrated that FBLN5 interacts with integrins and influences RAC1 activity, which is crucial for actin remodeling in Schwann cells during myelination .

How can FBLN5 antibody be utilized in studying peripheral nervous system myelination?

Research has identified FBLN5 as a critical factor in peripheral nervous system myelination, particularly in Schwann cell development and function . Methods for studying this role include:

• Co-culture systems: WJ-MSCs and Schwann cells can be co-cultured to study FBLN5 secretion and its effects on Schwann cell proliferation. ELISA using biotin-conjugated FBLN5 antibodies can quantify secretion levels, which have been shown to increase from 12.5 ± 0.99 pg/mL in single culture to 18.41 ± 0.26 pg/mL in co-culture conditions .

• Recombinant FBLN5 treatment: Purified recombinant FBLN5 can be applied to Schwann cell cultures to assess proliferation through ERK activation, mimicking the paracrine effects observed in co-culture systems .

• FBLN5 depletion studies: Knockdown of FBLN5 in WJ-MSCs using siRNA or CRISPR-Cas9 can help establish causality in observed effects on Schwann cell development .

• Actin remodeling assessment: Visualization of lamellipodia and filopodia formation in Schwann cells using fluorescent phalloidin staining following FBLN5 treatment can reveal its effects on cytoskeletal reorganization necessary for myelination .

• RAC1 activity assays: As FBLN5 modulates actin remodeling through RAC1 activity, GTPase activity assays can quantify changes in RAC1 activation following FBLN5 treatment .

• In vivo myelination models: Zebrafish models of Charcot-Marie-Tooth disease type 1 have been used to demonstrate FBLN5's ability to restore myelination defects in Schwann cells .

What are the recommended protocols for using biotin-conjugated FBLN5 antibody in ELISA?

For optimal ELISA performance with biotin-conjugated FBLN5 antibody, the following protocol considerations are recommended:

• Plate preparation: Coat high-binding ELISA plates with capture antibody (typically an unconjugated anti-FBLN5 antibody) at optimal concentration determined through titration.

• Blocking: Use appropriate blocking buffer (typically BSA or serum-based) to minimize non-specific binding.

• Sample preparation: Carefully prepare samples (cell culture media, tissue lysates, serum) with consideration for potential interfering substances.

• Biotin-conjugated antibody application: Apply biotin-conjugated FBLN5 antibody as detection antibody, with optimal dilution determined experimentally for each lot and application.

• Signal development: Use streptavidin-HRP followed by appropriate substrate for colorimetric detection.

• Standard curve generation: Include a standard curve using recombinant FBLN5 protein for accurate quantification.

• Controls: Include positive controls (samples known to contain FBLN5), negative controls, and blank wells for background assessment.

The exact dilutions should be determined by individual researchers through titration experiments, as optimal concentrations will depend on the specific experimental system .

How can researchers effectively use FBLN5 antibodies in disease models of myelin-related disorders?

FBLN5 has shown promise in addressing myelination defects in models of demyelinating diseases, particularly Charcot-Marie-Tooth disease . Effective use of FBLN5 antibodies in such models includes:

• Disease model characterization: Clear baseline characterization of myelination defects in the model using histological and functional assessments.

• Intervention protocols: Treatment with recombinant FBLN5 or FBLN5-producing stem cells at optimal time points in disease progression.

• Biodistribution tracking: Using labeled FBLN5 antibodies to track the distribution and persistence of administered FBLN5 in treated tissues.

• Mechanism investigation: Combining FBLN5 treatment with inhibitors of downstream pathways (e.g., RAC1 inhibitors) to confirm mechanistic hypotheses.

• Functional recovery assessment: Comprehensive evaluation of functional outcomes following FBLN5 treatment, correlating with molecular and cellular changes.

• Comparison with standard therapies: Benchmarking FBLN5-based approaches against current standard treatments for myelin disorders.

Research has demonstrated that FBLN5 effectively restores myelination defects in Schwann cells in zebrafish models of Charcot-Marie-Tooth disease type 1 through binding to Integrin and modulating actin remodeling via RAC1 activity .

What are the technical considerations for using FBLN5 antibodies in immunohistochemistry of neural tissues?

For effective immunohistochemistry of neural tissues using FBLN5 antibodies, researchers should consider:

• Tissue fixation and processing: Neural tissues require careful fixation to preserve both tissue architecture and antigenicity of FBLN5 epitopes.

• Antigen retrieval optimization: For FBLN5 detection, antigen retrieval with TE buffer pH 9.0 is suggested, with citrate buffer pH 6.0 as an alternative .

• Antibody dilution range: For unconjugated FBLN5 antibodies, a dilution range of 1:50-1:500 is recommended for IHC, but this should be optimized for each specific tissue and experimental condition .

• Control tissues: Include positive control tissues such as mouse lung tissue, mouse colon tissue, human lung cancer tissue, and human colon cancer tissue, where FBLN5 expression has been confirmed .

• Background reduction: Implement appropriate blocking steps to minimize non-specific binding, particularly in neural tissues which can exhibit high background.

• Double-labeling techniques: Consider co-staining with markers for Schwann cells, neurons, or myelin to contextualize FBLN5 expression within the neural architecture.

• Quantification methods: Develop consistent quantification protocols for immunohistochemical staining to allow for reliable comparisons between experimental groups.

How is FBLN5 involved in peripheral nervous system disorders, and how can antibodies help investigate these mechanisms?

FBLN5 plays a significant role in peripheral nervous system disorders, particularly those involving myelination defects. Recent research has illuminated several mechanisms:

• Schwann cell proliferation: FBLN5 promotes Schwann cell proliferation through ERK activation, a process that can be disrupted in various neuropathies .

• Integrin-mediated signaling: FBLN5 binds to Integrin receptors on Schwann cells, activating downstream signaling cascades essential for proper myelination .

• Actin cytoskeleton remodeling: Through RAC1 activation, FBLN5 modulates the formation of lamellipodia and filopodia in Schwann cells, structures necessary for myelin ensheathment of axons .

• Restoration of compact myelination: In models of Charcot-Marie-Tooth disease, FBLN5 has been shown to restore compact myelination of diseased axons by promoting actin remodeling mechanisms .

FBLN5 antibodies can be instrumental in investigating these mechanisms through:

• Immunolocalization of FBLN5 in healthy versus diseased peripheral nerves

• Blocking experiments to inhibit specific FBLN5-integrin interactions

• Quantification of FBLN5 expression levels in various neuropathological conditions

• Tracking therapeutic FBLN5 delivery in experimental treatment approaches

What are the methodological approaches for studying the relationship between FBLN5 and mesenchymal stem cell therapies?

The relationship between FBLN5 and mesenchymal stem cell therapies, particularly using Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs), can be studied through several methodological approaches:

• Secretome analysis: Characterizing the secretome of WJ-MSCs using mass spectrometry and ELISA to quantify FBLN5 secretion under various conditions. Co-culture experiments have shown that WJ-MSCs secrete higher levels of FBLN5 (18.41 ± 0.26 pg/mL) when cultured with Schwann cells compared to when cultured alone (12.5 ± 0.99 pg/mL) .

• Genetic modification approaches: Creating FBLN5-overexpressing or FBLN5-depleted WJ-MSCs to study the specific contribution of this factor to the therapeutic effects of stem cells .

• Comparative efficacy studies: Comparing the therapeutic efficacy of WJ-MSCs versus purified recombinant FBLN5 in models of peripheral neuropathy.

• Mechanisms of action investigations: Using pathway inhibitors to block specific signaling cascades (e.g., ERK, RAC1) to determine the precise mechanisms by which FBLN5 mediates the therapeutic effects of WJ-MSCs .

• Delivery optimization: Developing and testing various delivery methods for both WJ-MSCs and FBLN5 to maximize therapeutic potential in peripheral nerve disorders.

• Long-term efficacy and safety: Assessing the duration of therapeutic effects and potential adverse events associated with both WJ-MSC and FBLN5 treatments.

This research has significant implications for developing stem cell-based or FBLN5-based therapies for myelin-related diseases, including Charcot-Marie-Tooth disease .