FBLN5 Antibody

What is FBLN5 and why is it important in biological research?

FBLN5 (Fibulin-5) is a 448-amino acid protein with an expected molecular weight of approximately 50.2 kDa that plays an essential role in elastic fiber formation and organization. It functions as a critical mediator in the assembly of continuous elastin polymers and promotes interactions between microfibrils and elastin. FBLN5 stabilizes and organizes elastic fibers in multiple tissues including skin, lung, and vasculature. The protein also promotes adhesion of endothelial cells through interactions with integrin receptors via its RGD motif, potentially contributing to vascular development and remodeling. FBLN5 may function as an adapter molecule mediating interactions between fibrillin-1 (FBN1) and elastin (ELN), making it a crucial component of the extracellular matrix scaffold. Research on FBLN5 is particularly important because mutations in this protein have been linked to connective tissue disorders like cutis laxa and age-related macular degeneration, offering insights into pathological mechanisms and potential therapeutic targets.

What alternative names and characteristics should researchers be aware of when working with FBLN5?

When researching or ordering FBLN5 antibodies, it's important to recognize the various alternative designations for this protein to ensure comprehensive literature searches and appropriate reagent selection. FBLN5 may also be referenced as ADCL2, DANCE (Developmental arteries and neural crest EGF-like protein), ARMD3, ARCL1A, Urine p50 protein (UP50), or FIBL-5 in scientific literature and antibody catalogs. The human protein has a reported amino acid length of 448 and an expected molecular mass of 50.2 kDa, providing important reference points for experimental validation. Researchers should note that FBLN5 is evolutionarily conserved across species, with variants found in canine, porcine, monkey, mouse, and rat models, facilitating comparative studies and the use of animal models. Understanding these nomenclature variations is critical for comprehensive literature searches and experimental design.

What are the most common research applications for FBLN5 antibodies?

FBLN5 antibodies serve multiple experimental purposes across diverse research applications. Based on available commercial antibodies, the most common applications include:

• Western blotting (WB): For detecting and quantifying FBLN5 protein in tissue or cell lysates, with expected bands around 50.2 kDa.

• Immunohistochemistry (IHC): Used on paraffin-embedded tissue sections to visualize FBLN5's tissue distribution and localization, particularly in relation to elastic fibers.

• Immunocytochemistry/Immunofluorescence (ICC/IF): For examining subcellular localization and distribution patterns in cultured cells.

• Enzyme-linked immunosorbent assay (ELISA): For quantitative determination of FBLN5 in biological samples.

• Immunoprecipitation (IP): For isolating FBLN5 protein complexes to study protein-protein interactions, particularly relevant given FBLN5's adapter role between elastin and microfibrils.

Many commercial antibodies are validated for multiple applications, with monoclonal antibodies offering high specificity for particular epitopes and polyclonal antibodies providing broader epitope recognition. Researchers should select antibodies based on their specific experimental requirements and the validated applications listed by manufacturers.

How should I select the most appropriate FBLN5 antibody for studying specific protein domains or post-translational modifications?

Selecting the optimal FBLN5 antibody for studying specific domains or post-translational modifications requires careful consideration of several factors. First, examine the epitope specificity of available antibodies, as some target specific regions like the C-terminus (such as the anti-Fibulin 5 antibody [C3], C-term) while others recognize middle regions or N-terminal domains. For domain-specific studies, choose antibodies raised against the precise region of interest—for example, when investigating integrin interactions, select antibodies that don't interfere with the RGD motif.

Consider the antibody format—monoclonal antibodies offer high specificity for a single epitope, making them ideal for studying specific domains, while polyclonal antibodies recognize multiple epitopes and provide more robust general detection. Review available validation data thoroughly, including Western blots demonstrating specificity and cross-reactivity profiles. The antibody format should align with your experimental design—unconjugated antibodies for most applications, while conjugated versions (with fluorophores, enzymes, or biotin) may be preferable for specific detection methods.

For post-translational modifications, specialized antibodies that specifically recognize modified forms may be available, or you may need to combine immunoprecipitation using general FBLN5 antibodies with subsequent analysis of modifications using mass spectrometry or modification-specific detection methods. Always verify species reactivity matches your experimental model—many FBLN5 antibodies are available with reactivity to human, mouse, and rat proteins.

What experimental considerations are essential when investigating FBLN5's role in elastic fiber assembly?

Investigating FBLN5's role in elastic fiber assembly requires specialized experimental approaches addressing both molecular interactions and structural organization. When designing co-immunoprecipitation experiments to study FBLN5's interactions with elastin and microfibrils, use non-denaturing conditions that preserve native protein complexes. Since FBLN5 may act as an adapter mediating interactions between fibrillin-1 and elastin, include antibodies against these proteins as well.

For immunofluorescence studies, optimize fixation protocols to preserve elastic fiber architecture—standard formalin fixation may be suitable, but specialized fixatives may better maintain elastic fiber integrity. Co-localization experiments should include markers for other elastic fiber components (elastin, fibrillin-1) to confirm FBLN5's association with these structures. Super-resolution microscopy can provide more detailed insights into FBLN5's spatial arrangement within elastic fibers than conventional microscopy.

In functional studies, consider comparing elastic fiber assembly in cell cultures from normal individuals versus those with FBLN5 mutations. Time-course experiments can track the temporal sequence of FBLN5 incorporation into developing elastic fibers. For mechanical testing, atomic force microscopy or other biophysical approaches can assess how FBLN5 deficiency or mutation affects elastic fiber physical properties.

Include appropriate controls in all experiments—positive controls from tissues known to express FBLN5 abundantly (blood vessels, skin, lung) and negative controls from tissues with minimal expression or from FBLN5-knockdown models. These comprehensive approaches will provide robust insights into FBLN5's critical role in elastic fiber biology.

How can FBLN5 antibodies be leveraged to investigate genetic variants associated with disease?

FBLN5 antibodies provide powerful tools for investigating disease-associated genetic variants, particularly the ten distinct heterozygous missense mutations linked to age-related macular degeneration and the homozygous mutations (p.C217R, p.S227P, and R284X) associated with cutis laxa. For comparing wild-type and mutant FBLN5 expression patterns, immunohistochemistry with well-validated antibodies can reveal differences in protein localization, abundance, or aggregation in patient-derived tissues or engineered cell models expressing disease variants.

Western blot analysis using FBLN5 antibodies can assess whether mutations affect protein stability, processing, or secretion, potentially revealing mechanisms underlying pathology. For mutations that might affect specific domains, epitope-specific antibodies can determine if particular functions are compromised while others remain intact. In cases where mutations may alter FBLN5's interactions with binding partners, co-immunoprecipitation with FBLN5 antibodies followed by detection of interaction partners can identify disrupted molecular relationships.

For functional studies, FBLN5 antibodies can be used in cell culture models to compare elastic fiber assembly capabilities between wild-type and mutant forms. Researchers should consider developing or obtaining antibodies specifically recognizing common disease-associated mutations, which would enable direct detection of mutant protein in heterozygous samples. When working with patient-derived materials, combining genetic analysis with FBLN5 immunodetection provides powerful correlations between genotype and protein phenotype, potentially revealing mechanisms underlying variable disease expressivity.

What are the optimal conditions for using FBLN5 antibodies in Western blotting experiments?

Optimizing Western blot protocols for FBLN5 detection requires attention to several key parameters. For sample preparation, use RIPA or NP-40 based lysis buffers containing protease inhibitors to prevent degradation of this extracellular matrix protein. When analyzing secreted FBLN5, collect and concentrate cell culture media using methods that preserve protein integrity. For tissue samples, specialized extraction protocols may be needed to efficiently solubilize matrix-associated proteins.

During gel electrophoresis, use 8-12% SDS-PAGE gels appropriate for resolving FBLN5's expected 50.2 kDa molecular weight. Transfer proteins to PVDF or nitrocellulose membranes using wet transfer systems, which often work better for extracellular matrix proteins than semi-dry methods. For blocking, 5% non-fat dry milk or BSA in TBST is typically effective, with overnight blocking at 4°C potentially reducing background for some antibodies.

Primary antibody dilutions vary by manufacturer (typically 1:500-1:2000), so consult specific product datasheets, but overnight incubation at 4°C generally yields optimal results. Use appropriate HRP-conjugated secondary antibodies (typically 1:5000-1:10000) matched to your primary antibody species, incubating for 1-2 hours at room temperature. For detection, standard ECL systems are usually sufficient, but more sensitive detection methods may be beneficial for low-abundance samples.

Always include appropriate controls: positive controls from tissues known to express FBLN5 (vascular tissues are often good choices), negative controls (tissues with minimal expression), and loading controls such as β-actin or GAPDH. For troubleshooting, common issues include weak or absent signal (try increased antibody concentration or longer incubation) and high background (optimize blocking and washing conditions).

What considerations are important for successful immunohistochemical detection of FBLN5?

Successful immunohistochemical detection of FBLN5 in tissues requires optimization of multiple parameters. For fixation, 10% neutral buffered formalin is generally suitable, but fixation time should be standardized (typically 24-48 hours) to maintain consistent results. Paraffin-embedded sections typically work well, but for certain applications, frozen sections may better preserve antigenicity. Section thickness of 4-5 μm is standard for most applications.

Antigen retrieval is often critical—heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) are good starting points, but optimal conditions may vary depending on the specific antibody and tissue type. For tissues rich in extracellular matrix, additional optimization of retrieval conditions may be necessary to expose FBLN5 epitopes within dense matrix structures.

For blocking, use 5-10% normal serum (from the same species as the secondary antibody) or commercial blocking solutions to reduce non-specific binding. Primary antibody dilutions typically range from 1:100-1:500 for IHC, with overnight incubation at 4°C often yielding optimal results. Detection systems can include standard ABC or polymer-based detection methods, with DAB as a common chromogen, though fluorescent secondary antibodies may provide better resolution for co-localization studies.

Always include appropriate controls: positive control tissues known to express FBLN5 (vascular tissues, skin, or lung are good choices), negative controls (primary antibody omission or isotype controls), and when possible, tissues from FBLN5-deficient models. For tissues where FBLN5 may be associated with elastic fibers, consider counterstaining with elastin-specific stains like Verhoeff-Van Gieson to confirm appropriate localization patterns.

How can I optimize co-immunoprecipitation protocols for studying FBLN5 protein interactions?

Optimizing co-immunoprecipitation (co-IP) protocols for studying FBLN5 protein interactions requires careful consideration of multiple factors to preserve physiologically relevant interactions. Begin with lysis buffer selection—use non-denaturing buffers (e.g., containing 1% NP-40 or Triton X-100) with protease inhibitors to maintain protein complexes while effectively solubilizing membrane-associated proteins. For extracellular matrix proteins like FBLN5, specialized extraction buffers may be necessary to release proteins from matrix associations.

Choose antibodies specifically validated for immunoprecipitation applications—several commercial FBLN5 antibodies are validated for IP as indicated in the search results. For optimal results, use 2-5 μg of antibody per 500 μg of total protein lysate. Pre-clear lysates with protein A/G beads to reduce non-specific binding, which is particularly important when working with complex tissue samples.

For co-IP of weakly associated partners, consider using reversible cross-linking reagents before cell lysis to stabilize transient interactions. Incubate lysates with FBLN5 antibodies overnight at 4°C with gentle rotation, followed by addition of protein A/G beads for 1-4 hours. Use multiple gentle washes with buffer containing lower detergent concentrations than the lysis buffer to remove non-specifically bound proteins while preserving genuine interactions.

Include critical controls: negative control IPs using isotype-matched control antibodies, input samples representing pre-IP lysate, and where possible, reverse co-IPs where antibodies against suspected interaction partners are used for precipitation followed by FBLN5 detection. Based on FBLN5's known functions, elastin, fibrillin-1, and integrin family members would be logical interaction partners to investigate. For unbiased identification of novel interaction partners, mass spectrometry analysis of co-immunoprecipitated proteins can be highly informative.

How do I interpret variable staining patterns observed in FBLN5 immunohistochemistry?

Interpreting variable FBLN5 staining patterns requires careful consideration of both biological and technical factors. Biologically, FBLN5 expression naturally varies across tissues and developmental stages—it's highly expressed in elastic fiber-rich tissues like blood vessels, skin, and lung, with distinct localization patterns reflecting its role in elastic fiber organization. Age-related changes in elastic fiber content and organization may also influence staining patterns, potentially complicating comparisons between samples from subjects of different ages.

Disease states can significantly alter FBLN5 expression or localization—mutations associated with age-related macular degeneration or cutis laxa may affect protein levels, distribution, or antibody recognition. In such cases, variability may represent genuine biological differences rather than technical artifacts. FBLN5's distribution may also vary within a single tissue, with concentration in specific structures like blood vessel walls or elastic laminae.

Technically, variable staining may result from inconsistent fixation, processing, or antigen retrieval conditions. Standardize these parameters and consider automated staining platforms for improved consistency. Antibody-related factors including lot-to-lot variability, degradation of stored antibodies, or differences in affinity for various FBLN5 epitopes can contribute to inconsistent results. Testing multiple antibodies targeting different FBLN5 epitopes can help distinguish genuine expression patterns from technical artifacts.

When evaluating staining patterns, consider both intensity and distribution—FBLN5 typically shows extracellular, fibrillar patterns consistent with elastic fiber association. Unexpected patterns like nuclear or cytoplasmic staining should be interpreted cautiously and validated with additional techniques. Correlation with elastic fiber-specific stains or co-staining with other elastic fiber components can confirm whether FBLN5 localization aligns with its expected biological context.

What approaches can help differentiate true FBLN5 signal from non-specific binding in complex tissues?

Differentiating true FBLN5 signal from non-specific binding requires a multi-faceted validation approach. Proper controls are essential—include negative controls omitting primary antibody, using isotype-matched control antibodies at the same concentration, or using pre-immune serum to establish background staining levels. Antibody absorption controls, where FBLN5 antibody is pre-incubated with recombinant FBLN5 protein to block specific binding sites, can confirm signal specificity.

Validation across multiple techniques strengthens confidence in observed results. Verify antibody specificity by Western blot, confirming a single band at the expected 50.2 kDa molecular weight. Correlation of immunostaining patterns with FBLN5 mRNA expression (using in situ hybridization or RT-PCR) provides complementary evidence of specificity. Using multiple antibodies targeting different FBLN5 epitopes can help confirm consistent staining patterns independent of the specific epitope recognized.

Staining pattern evaluation provides further validation—specific FBLN5 staining should correlate with expected localization patterns. Based on FBLN5's biology, expect extracellular matrix localization, particularly associated with elastic fibers in tissues like blood vessels, skin, and lung. Non-specific staining often shows unusual patterns inconsistent with known biology, such as uniform staining across all tissue types regardless of expected expression differences.

Co-localization studies provide powerful validation—FBLN5 should co-localize with other elastic fiber components. Dual staining with elastin or fibrillin-1 antibodies helps confirm specific FBLN5 labeling consistent with its biological function. Titration experiments can also distinguish specific from non-specific signal, as specific staining typically persists at higher antibody dilutions while background diminishes.

How should I reconcile differences between FBLN5 protein levels detected by Western blot versus immunohistochemistry?

Reconciling differences between FBLN5 protein levels detected by Western blot versus immunohistochemistry requires understanding the inherent differences between these techniques. Methodologically, Western blot detects denatured protein separated by size, while IHC visualizes proteins in their tissue context with more native conformations. This fundamental difference means epitope accessibility may vary significantly between techniques—some epitopes may be masked in tissue sections but exposed after denaturation, or vice versa.

Antibody considerations are critical—different antibodies may be used for each technique, recognizing different epitopes with varying affinities. Some antibodies work exceptionally well for Western blot but poorly for IHC, or the reverse. Check whether the same antibody was used for both applications and whether it's validated for both. If different antibodies were used, they may have different sensitivities or recognize different forms of the protein.

Sample preparation effects substantially impact results—FBLN5, as an extracellular matrix protein, may be difficult to fully extract for Western blot, potentially underestimating actual abundance. Conversely, fixation for IHC may mask epitopes, reducing detection sensitivity. FBLN5's association with elastic fibers may make quantitative extraction particularly challenging, as these structures are often resistant to solubilization.

Biological considerations also explain apparent discrepancies—FBLN5 distribution is spatially heterogeneous within tissues, concentrated in elastic fiber-rich regions. IHC reveals this spatial heterogeneity, while Western blot averages protein content across the entire sample. A tissue with focal areas of high FBLN5 expression may show strong IHC staining in specific regions but moderate signal by Western blot due to dilution effect.

For accurate interpretation, use complementary techniques like ELISA or mass spectrometry as additional reference points. Include appropriate positive and negative controls in both methods, and consider mRNA expression analysis to provide another perspective on FBLN5 expression levels.

How can FBLN5 antibodies help elucidate mechanisms of age-related macular degeneration?

FBLN5 antibodies provide powerful tools for investigating age-related macular degeneration (AMD) mechanisms, particularly given the association of FBLN5 mutations with this disease. For expression analysis, comparing FBLN5 levels and distribution patterns between normal and AMD-affected retinal tissues using immunohistochemistry can reveal whether alterations in FBLN5 expression or localization correlate with disease progression. Western blot analysis can quantify potential changes in FBLN5 protein levels or processing in AMD versus control samples.

Mutation-specific studies are particularly relevant given the ten heterozygous FBLN5 missense mutations associated with AMD mentioned in the search results. Researchers can use site-directed mutagenesis to create cell models expressing these specific mutations, then employ FBLN5 antibodies to assess how these mutations affect protein localization, secretion, or stability. Comparing the behavior of wild-type versus mutant FBLN5 in retinal pigment epithelium cell culture models can provide insights into pathogenic mechanisms.

For structural studies, immunogold electron microscopy using FBLN5 antibodies can examine ultrastructural changes in Bruch's membrane organization in AMD, potentially revealing how FBLN5 mutations contribute to the accumulation of drusen or other AMD-associated deposits. Co-localization studies combining FBLN5 antibodies with markers for oxidative damage can investigate potential relationships between FBLN5 dysfunction and oxidative stress, a key factor in AMD pathogenesis.

In therapeutic development contexts, FBLN5 antibodies can monitor changes in protein expression or localization in response to experimental treatments, helping evaluate whether interventions successfully restore normal FBLN5 function or compensate for its dysfunction. As AMD is a complex, multifactorial disease, combining FBLN5 studies with investigations of other AMD-associated molecules will likely provide the most comprehensive understanding of disease mechanisms.

What differences should researchers expect when using FBLN5 antibodies to study cutis laxa versus age-related macular degeneration?

Researchers using FBLN5 antibodies to study these distinct FBLN5-associated disorders should expect several important differences in experimental approaches and findings. In terms of mutation effects, cutis laxa typically involves homozygous FBLN5 mutations (p.C217R, p.S227P, and R284X) causing severe loss of function, while AMD involves heterozygous missense mutations with likely subtler effects on protein function. This fundamental difference means antibody detection may reveal more dramatic alterations in cutis laxa samples—potentially showing complete absence or severely reduced FBLN5 in homozygous mutation cases.

Tissue distribution considerations are crucial—cutis laxa affects multiple elastic fiber-rich tissues (skin, lung, vasculature), while AMD primarily affects ocular tissues. Researchers should adapt their experimental protocols accordingly, with cutis laxa studies focusing on skin biopsies, vascular samples, or lung tissue, while AMD research requires specialized techniques for ocular tissues like retinal pigment epithelium and choroid.

Developmental timing differs significantly—cutis laxa is often congenital or early-onset, suggesting FBLN5 antibody studies should include developmental time points when elastic fibers are being assembled. AMD is age-related, indicating that studies should incorporate aging models and examine how FBLN5 function changes over time. This temporal difference influences experimental design, with cutis laxa research potentially focusing on developmental processes and AMD research on age-related changes and cumulative damage.

Functional assays would also differ—cutis laxa studies might emphasize elastic fiber assembly and mechanical properties of affected tissues, while AMD research would focus more on Bruch's membrane integrity, RPE-choroid interactions, and oxidative stress responses. While both conditions involve FBLN5 dysfunction, the distinct pathogenic mechanisms require tailored experimental approaches utilizing FBLN5 antibodies in context-appropriate ways.

How can FBLN5 antibodies contribute to developing therapeutic strategies for elastic fiber disorders?

FBLN5 antibodies play crucial roles in developing therapeutic strategies for elastic fiber disorders through multiple research applications. For therapeutic target validation, immunostaining with FBLN5 antibodies in disease models and patient samples helps establish whether FBLN5 dysfunction is a primary pathogenic mechanism or secondary consequence, informing whether directly targeting FBLN5 would be beneficial. Western blot analysis using these antibodies can quantify changes in FBLN5 expression, processing, or degradation in response to experimental therapeutics.

In gene therapy development, FBLN5 antibodies enable monitoring of protein expression after gene delivery, confirming successful restoration of FBLN5 in deficient tissues. This application is particularly relevant for conditions like cutis laxa involving homozygous loss-of-function mutations. For protein replacement therapies, these antibodies help track the distribution, stability, and incorporation of administered recombinant FBLN5 into existing matrix structures.

FBLN5 antibodies facilitate high-throughput screening assays for small molecule discovery, enabling development of compound libraries that enhance FBLN5 stability, secretion, or function. Such assays might identify chaperones that help mutant FBLN5 fold correctly or compounds that enhance residual function of partially defective protein. For cell-based therapies, these antibodies verify that engineered cells produce functional FBLN5 before and after transplantation.

In biomarker development, FBLN5 antibodies can be incorporated into assays detecting FBLN5 fragments or abnormal forms in blood or other accessible fluids, potentially providing non-invasive disease monitoring tools. For personalized medicine approaches, these antibodies help characterize how specific FBLN5 mutations affect protein function, potentially guiding mutation-specific therapeutic strategies.

By supporting these diverse research applications, FBLN5 antibodies constitute essential tools for advancing therapeutic development for elastic fiber disorders, from mechanistic understanding to clinical translation.