FBN2 Antibody, Biotin conjugated

What is FBN2 and why is it an important target for antibody-based research?

Fibrillin 2 (FBN2) is a secreted protein with a canonical length of 2912 amino acid residues and a molecular mass of approximately 314.8 kDa in humans. As a member of the Fibrillin protein family, FBN2 plays crucial roles in eye development and carbohydrate metabolism/homeostasis. The protein is notably expressed in the placenta and exists in up to two different isoforms .

FBN2 has garnered research attention due to its importance as a marker for specific neuronal cell types, including Cerebral Cortex MGE Interneurons, Gray Matter MGE Interneurons, Brain Chandelier Neurons, and Gray Matter Chandelier Neurons. This makes FBN2 antibodies valuable tools for neuroscience research focused on cellular identification and characterization .

The protein undergoes several post-translational modifications, including O-glycosylation and N-glycosylation, which can affect antibody binding and experimental outcomes. Alternative names for this target include DA9, EOMD, fibrillin 5, and CCA, which researchers should be aware of when reviewing literature .

What are the key differences between biotin-conjugated and unconjugated FBN2 antibodies?

Biotin-conjugated FBN2 antibodies offer several advantages over their unconjugated counterparts, particularly in detection sensitivity and experimental flexibility. The biotin-streptavidin system provides one of the strongest non-covalent biological interactions known, with a dissociation constant (Kd) in the order of 10^-15 M, making these conjugates excellent for signal amplification in various assays .

Unconjugated antibodies require a secondary detection system, whereas biotin-conjugated antibodies can be directly detected using streptavidin or avidin coupled to various reporter molecules (enzymes, fluorophores, gold particles). This reduces the number of incubation steps and can minimize background signal in complex experimental systems .

Methodologically, biotin-conjugated antibodies are particularly advantageous in multiplex analysis and multicolor imaging applications. They allow for flexible experimental design where the same primary antibody can be used with different detection systems depending on the experimental requirements .

What experimental applications are best suited for FBN2 antibodies?

FBN2 antibodies demonstrate utility across multiple experimental applications, with varying levels of validation across suppliers. The most commonly validated applications include:

• Western Blot (WB): Widely validated across numerous suppliers, Western blotting allows for size-based confirmation of FBN2 detection and quantification. Given the large size of FBN2 (314.8 kDa), researchers should use low-percentage gels (4-8%) for optimal resolution .

• Enzyme-Linked Immunosorbent Assay (ELISA): Another commonly validated application for FBN2 antibodies. This method is particularly useful for quantitative analysis of FBN2 in biological samples .

• Immunofluorescence (IF): Several FBN2 antibodies are validated for immunofluorescence applications, making them suitable for localization studies in tissues and cells .

• Immunohistochemistry (IHC): Some antibodies are validated for IHC, allowing for visualization of FBN2 distribution in tissue sections .

• Immunoprecipitation (IP): Select antibodies are validated for immunoprecipitation, enabling the isolation of FBN2 and associated protein complexes .

For biotin-conjugated antibodies specifically, they excel in applications requiring signal amplification or multiplex analysis, including fluorescence-based plate assays (FLISA), multicolor imaging, and various commercial multiplex platforms .

What controls should be included when using FBN2 antibodies in experimental designs?

A robust experimental design utilizing FBN2 antibodies should incorporate several types of controls:

• Positive controls: Samples known to express FBN2, such as placental tissue or cell lines with confirmed FBN2 expression. This validates that the detection system works properly .

• Negative controls: Samples known not to express FBN2 or samples where FBN2 has been knocked down/out using siRNA or CRISPR-Cas9. This confirms specificity of the antibody .

• Isotype controls: Particularly important for flow cytometry and immunofluorescence applications to control for non-specific binding of antibodies based on their isotype characteristics rather than their antigen specificity.

• Absorption controls: Pre-incubation of the antibody with purified antigen should abolish specific staining, confirming antibody specificity.

• Secondary antibody-only controls: For experiments using unconjugated primary antibodies, this control helps identify background caused by the secondary detection system.

For biotin-conjugated antibodies specifically, additional controls should include samples treated with streptavidin/avidin alone to identify tissues with endogenous biotin, which could lead to false-positive results .

How can cross-reactivity be minimized when using FBN2 antibodies in complex tissue samples?

Minimizing cross-reactivity when using FBN2 antibodies requires a multi-faceted approach:

• Antibody selection: Choose antibodies that have undergone extensive validation for specificity. Suppliers often provide cross-reactivity data against related proteins in the fibrillin family (e.g., FBN1, FBN3) and other structurally similar proteins .

• Blocking optimization: Use a combination blocking approach with both protein blockers (BSA, serum matching the host of the secondary antibody) and non-protein blockers (commercial blockers containing detergents and other components) to reduce non-specific binding .

• Antibody titration: Determine the optimal antibody concentration through careful titration experiments. The ideal concentration provides the best signal-to-noise ratio without increasing background .

• Sample pre-absorption: For tissues with high levels of endogenous biotin (such as liver, kidney, and brain), pre-treat samples with avidin or streptavidin followed by biotin to block endogenous biotin before applying biotin-conjugated antibodies .

• Epitope-specific antibodies: Consider using antibodies targeting unique epitopes of FBN2 that are not conserved in related proteins. Immunoaffinity chromatography-purified antibodies, such as those prepared using antigen-coupled agarose beads followed by solid phase adsorption, show reduced cross-reactivity .

What are the optimal strategies for multiplexing FBN2 detection with other neuronal markers?

Effective multiplexing strategies for FBN2 with other neuronal markers require careful planning:

• Antibody compatibility: When multiplexing, select antibodies raised in different host species to enable discrimination using species-specific secondary antibodies. For instance, combine rabbit anti-FBN2 with mouse antibodies against other targets .

• Conjugate selection: Utilize biotin-conjugated FBN2 antibodies in conjunction with directly labeled antibodies against other markers. The biotin-streptavidin system can be combined with diverse fluorophores for multiplex fluorescence imaging .

• Sequential detection: For complex multiplexing, employ sequential detection protocols where primary and secondary antibodies for one marker are applied, followed by fixation, then detection of subsequent markers.

• Spectral unmixing: In advanced imaging systems, use spectral unmixing algorithms to separate overlapping fluorescent signals, allowing for more markers to be used simultaneously.

• Enzymatic detection systems: When using biotin-conjugated antibodies in colorimetric IHC multiplexing, select enzyme-substrate combinations that yield distinct, non-overlapping colors.

For neuronal subtype identification specifically, FBN2 has been validated as a marker for Cerebral Cortex MGE Interneurons, Gray Matter MGE Interneurons, Brain Chandelier Neurons, and Gray Matter Chandelier Neurons, making it valuable in neuronal classification studies when combined with other established markers .

How do post-translational modifications of FBN2 affect antibody binding and detection sensitivity?

Post-translational modifications (PTMs) of FBN2, particularly O-glycosylation and N-glycosylation, can significantly impact antibody recognition and experimental outcomes:

• Epitope masking: Glycosylation sites may physically block antibody access to protein epitopes, reducing binding efficiency. Researchers should select antibodies targeting epitopes known to be free from glycosylation sites .

• Conformational changes: PTMs can alter protein folding, changing the three-dimensional structure of epitopes even if they are distant from the modification site. This may enhance or reduce antibody binding depending on the specific antibody used.

• Sample preparation strategies: For comprehensive detection regardless of glycosylation state, researchers can employ enzymatic deglycosylation (using PNGase F for N-linked glycans or O-glycosidase for O-linked glycans) prior to antibody application.

• Modification-specific antibodies: For studies focusing on PTM status, specialized antibodies that specifically recognize glycosylated or non-glycosylated forms of FBN2 may be employed.

• Detection method selection: Western blotting can reveal multiple bands representing differently modified forms of FBN2, while immunofluorescence may show differential subcellular localization based on modification status .

Understanding the PTM profile of FBN2 in specific experimental contexts is crucial for accurate interpretation of antibody-based detection results and may provide insights into functional variations of the protein across different tissues and developmental stages.

What are the technical considerations for using biotin-conjugated FBN2 antibodies in super-resolution microscopy?

Super-resolution microscopy with biotin-conjugated FBN2 antibodies requires attention to several technical factors:

• Signal amplification optimization: While biotin-streptavidin systems provide excellent signal amplification, excessive amplification can compromise the spatial resolution in super-resolution techniques. Titration experiments should determine optimal concentration ratios .

• Probe size considerations: The complete detection system (primary antibody + biotin + streptavidin + fluorophore) creates a significant distance between the target epitope and the fluorescent signal. This "linkage error" must be accounted for in localization precision calculations.

• Sample preparation protocols: Super-resolution techniques require exceptional sample preparation. Use thin sections (≤10 μm), minimal fixation times, and permeabilization conditions that maintain antigen accessibility while preserving ultrastructure.

• Fluorophore selection: Choose fluorophores compatible with the specific super-resolution technique being used:

  • For STED: Select dyes with good depletion efficiency

  • For STORM/PALM: Use fluorophores with appropriate blinking characteristics

  • For SIM: Ensure fluorophores have high photostability and brightness

• Multi-color imaging strategy: When combining biotin-conjugated FBN2 antibodies with other markers in multi-color super-resolution, carefully select fluorophores with minimal spectral overlap and appropriate spatial distribution to facilitate computational separation .

By optimizing these parameters, researchers can effectively utilize biotin-conjugated FBN2 antibodies for high-precision localization studies of FBN2 in relation to other cellular structures at nanoscale resolution.

How can researchers troubleshoot inconsistent results when using FBN2 antibodies across different sample types?

Inconsistent results with FBN2 antibodies across sample types often stem from several key variables:

• Tissue/cell-specific expression patterns: FBN2 expression varies significantly across tissues, with notable expression in placenta but potentially different levels in other tissues. Researchers should verify expression levels through qPCR before antibody-based detection .

• Isoform variation: Up to two different isoforms of FBN2 have been reported, which may have different distributions and antibody accessibility depending on tissue type. Ensuring the selected antibody recognizes all relevant isoforms for the experimental context is essential .

• Fixation sensitivity: Optimize fixation protocols for each sample type. Some epitopes may be fixation-sensitive, requiring:

  • For frozen sections: Brief fixation with 2-4% paraformaldehyde

  • For paraffin sections: Antigen retrieval optimization

  • For cell cultures: Comparison of cross-linking vs. precipitating fixatives

• Biotin blocking requirements: Endogenous biotin levels vary dramatically across tissue types. Liver, kidney, and brain tissues typically require more rigorous blocking protocols when using biotin-conjugated antibodies or detection systems .

• Antibody concentration re-optimization: Each sample type may require different antibody concentrations. What works for cultured cells may be insufficient for tissue sections. Systematic titration experiments for each sample type should be conducted.

A methodical troubleshooting approach includes side-by-side comparison using multiple detection methods (IF, IHC, WB) on the same samples to identify whether inconsistencies are technique-dependent or sample-dependent.

How can FBN2 antibodies contribute to research on neurodevelopmental disorders?

FBN2 antibodies offer valuable tools for investigating neurodevelopmental processes and related disorders:

• Neuronal subtype identification: The validation of FBN2 as a marker for specific neuronal populations, including Cerebral Cortex MGE Interneurons and Brain Chandelier Neurons, makes FBN2 antibodies invaluable for studying interneuron development and function in both normal and pathological conditions .

• Developmental expression profiling: FBN2 is involved in eye development, suggesting temporal regulation during development. Antibodies can track expression changes across developmental stages, potentially revealing dysregulation in neurodevelopmental disorders.

• Circuit assembly studies: Biotin-conjugated FBN2 antibodies are particularly useful in multiplex immunofluorescence studies examining the integration of FBN2-expressing neurons into developing neural circuits, which may be altered in conditions like autism spectrum disorders.

• Post-mortem tissue analysis: In human post-mortem studies of neurodevelopmental disorders, FBN2 antibodies can identify alterations in specific neuronal populations that express this marker, potentially revealing cellular pathologies.

• Disease model validation: In animal or organoid models of neurodevelopmental disorders, FBN2 antibodies can help verify whether specific neuronal subtypes develop appropriately, providing insight into disease mechanisms.

The ability to specifically identify and analyze FBN2-expressing neuronal populations using well-characterized antibodies may reveal previously unrecognized connections between these cell types and neurodevelopmental pathologies.

What are the considerations for using FBN2 antibodies in high-throughput screening applications?

High-throughput screening with FBN2 antibodies requires specific optimization strategies:

• Assay miniaturization: Traditional ELISA formats using FBN2 antibodies can be adapted to 384- or 1536-well formats, but require careful optimization of antibody concentrations to maintain sensitivity while reducing reagent usage .

• Detection system selection: Biotin-conjugated FBN2 antibodies offer advantages for high-throughput applications due to the flexibility of detection systems. Streptavidin-conjugated reporters (fluorescent, chemiluminescent, or colorimetric) can be selected based on the available plate reader technology .

• Automation compatibility: Protocols should be developed with automation in mind, minimizing wash steps and incubation times where possible without compromising signal quality.

• Quality control parameters: Implement robust quality control measures, including:

  • Intra-plate positive and negative controls

  • Z'-factor calculation for assay validation

  • Coefficient of variation monitoring across plates and runs

• Multiplex potential: Biotin-conjugated FBN2 antibodies can be incorporated into multiplex screening platforms where multiple targets are assessed simultaneously, but require careful optimization to prevent cross-reactivity or signal interference .

Leveraging the specificity of FBN2 antibodies in high-throughput contexts can facilitate screening for compounds that modulate FBN2 expression or function, potentially identifying therapeutic candidates for conditions involving FBN2 dysregulation.