FBLN5 Antibody, HRP conjugated

What is FBLN5 and what are its key biological functions?

FBLN5, also known as Fibulin-5 (and alternatively as DANCE, ADCL2, ARMD3, and ARCL1A), is an extracellular matrix glycoprotein with a calculated molecular weight of approximately 50 kDa. It is secreted by various cell types including vascular smooth muscle cells, fibroblasts, and endothelial cells .

FBLN5 contains an Arg-Gly-Asp (RGD) motif and calcium-binding EGF-like domains that facilitate its interaction with cellular components . The protein serves several critical biological functions:

• Essential for elastic fiber formation and assembly of continuous elastin polymer

• Promotes interaction between microfibrils and elastin

• Stabilizes and organizes elastic fibers in the skin, lung, and vasculature

• Promotes adhesion of endothelial cells through interaction with integrins via its RGD motif

• Functions as a vascular ligand for integrin receptors

• May act as an adapter mediating interaction between fibrillin-1 (FBN1) and elastin (ELN)

FBLN5 dysregulation has been implicated in several pathological conditions, including age-related macular degeneration, Charcot-Marie-Tooth neuropathies, and vascular remodeling in atherosclerotic plaques .

What applications are validated for FBLN5 antibodies?

FBLN5 antibodies have been validated for multiple experimental applications across different research contexts. Based on comprehensive validation data, these antibodies demonstrate utility in:

• Western Blot (WB): Effective at dilutions ranging from 1:1000 to 1:8000

• Immunohistochemistry (IHC): Recommended dilutions between 1:50 and 1:500

• Immunoprecipitation (IP): Typically using 0.5-4.0 μg antibody for 1.0-3.0 mg of total protein lysate

• Immunofluorescence (IF): Successfully employed in visualization studies

• ELISA: Effective for quantitative detection

For HRP-conjugated FBLN5 antibodies specifically, these provide enhanced sensitivity for chromogenic detection without requiring secondary antibodies, streamlining experimental protocols and reducing background in sensitive applications.

The variety of validated applications makes these antibodies versatile tools for investigating FBLN5 in diverse experimental settings, from protein expression analysis to localization studies and protein-protein interaction investigations.

What is the expected molecular weight pattern for FBLN5 in Western blot analysis?

FBLN5 shows variability in its observed molecular weight in Western blot analysis, which is important for researchers to consider when interpreting their results. While the calculated molecular weight based on amino acid sequence is approximately 50 kDa (448 amino acids), the observed molecular weight typically ranges between 50-65 kDa .

Specifically, researchers may observe:

• A 50 kDa band corresponding to the unmodified protein

• A 55 kDa band representing partially modified protein

• A 65-66 kDa band representing the fully glycosylated form of the protein

This variability is primarily attributed to post-translational modifications, particularly glycosylation. The 66 kDa band specifically corresponds to fully glycosylated FBLN5 protein . These variations can also be tissue-dependent, as different tissues may process the protein differently.

When analyzing Western blot results, researchers should anticipate potential multiple bands and consider the glycosylation status of FBLN5 in their specific experimental system or tissue type.

How is FBLN5 expression regulated by hypoxia in endothelial cells?

FBLN5 has been identified as a hypoxia-responsive gene in endothelial cells, with significant implications for vascular biology research. Hypoxic conditions (typically 1-2% O₂) increase FBLN5 mRNA levels in endothelial cells in a time-dependent manner, with maximal induction (approximately 2.5-fold increase) achieved after 24 hours of hypoxia exposure .

The molecular mechanisms underlying this hypoxic regulation involve:

• Activation of the PI3K/Akt/mTOR signaling pathway, as evidenced by the ability of pathway inhibitors (LY294002 and rapamycin) to block hypoxia-induced FBLN5 upregulation

• HIF-1α (Hypoxia-Inducible Factor 1-alpha) dependent transcriptional activation, confirmed through silencing experiments and dimethyl oxalylglycine treatment

• Direct binding of HIF-1 to a hypoxia response element (HRE) located at -78 bp in the FBLN5 promoter, demonstrated through EMSA and ChIP assays

Functionally, this hypoxia-induced FBLN5 expression appears to contribute to endothelial cell survival, as FBLN5 knockdown increases the rate of apoptosis in hypoxia-exposed endothelial cells . This suggests that FBLN5 induction represents an adaptive survival mechanism in response to hypoxic stress.

For researchers investigating vascular responses to hypoxia, these findings highlight FBLN5 as a potential marker and mediator of endothelial adaptation to hypoxic conditions.

What experimental controls are essential when using FBLN5 antibodies?

When conducting experiments with FBLN5 antibodies, particularly HRP-conjugated versions, implementing proper controls is crucial for generating reliable and interpretable data. Essential experimental controls include:

• Positive tissue controls: Include verified FBLN5-expressing tissues such as:

  • Mouse: heart, brain, placenta, skeletal muscle, kidney tissues

  • Rat: brain and heart tissues

  • Human: heart and pancreas tissues

• Negative controls:

  • Primary antibody omission control

  • Isotype control (using matched IgG at the same concentration)

  • FBLN5 knockout or knockdown samples when available

• Loading controls: For Western blotting, include housekeeping proteins (β-actin, GAPDH) to normalize expression levels

• Molecular weight markers: Include appropriate weight standards to verify detection at expected molecular weights (50-65 kDa range)

• Antigen competition: Pre-absorb antibody with FBLN5 recombinant protein to confirm specificity

• Cross-reactivity assessment: When working with tissues from different species, verify antibody cross-reactivity

• HRP-specific controls:

  • Substrate-only control to assess endogenous peroxidase activity

  • Signal development time curves to determine optimal exposure conditions

Proper implementation of these controls helps validate antibody specificity, optimize experimental conditions, and ensure accurate interpretation of results, particularly important when investigating proteins like FBLN5 that undergo post-translational modifications affecting their molecular weight and detection patterns.

How can researchers distinguish between glycosylated and non-glycosylated forms of FBLN5?

Distinguishing between glycosylated and non-glycosylated forms of FBLN5 is essential for understanding its functional state in different biological contexts. Based on current research methodologies, several approaches can be implemented:

• Western blot mobility analysis:

  • The fully glycosylated form appears at approximately 65-66 kDa

  • Partially glycosylated forms appear around 55 kDa

  • The non-glycosylated form appears at approximately 50 kDa (the calculated molecular weight)

• Enzymatic deglycosylation treatment:

  • Treat protein samples with glycosidases such as PNGase F (removes N-linked glycans) or O-glycosidase (removes O-linked glycans)

  • Compare mobility shift before and after treatment on Western blots

  • A shift from 65-66 kDa to 50 kDa after treatment confirms glycosylation

• Lectin-based detection methods:

  • Use lectins with specificity for different glycan structures in conjunction with FBLN5 antibodies

  • This approach can identify the presence and type of glycosylation

• Mass spectrometry analysis:

  • Provides detailed characterization of glycosylation sites and structures

  • Can be performed on immunoprecipitated FBLN5 using the antibody (0.5-4.0 μg for 1.0-3.0 mg of total protein)

• Glycosylation site mutants:

  • In experimental systems, mutations at predicted N-glycosylation sites can be introduced

  • Compare the mobility of wild-type and mutant proteins

These approaches allow researchers to determine the glycosylation status of FBLN5 in different tissues or experimental conditions, providing insights into how post-translational modifications might influence its function in various physiological and pathological contexts.

What are the recommended antigen retrieval methods for FBLN5 immunohistochemistry?

Successful immunohistochemical detection of FBLN5 requires appropriate antigen retrieval methods to expose epitopes that may be masked during fixation. Based on validated protocols, the following antigen retrieval approaches are recommended:

• Heat-induced epitope retrieval (HIER):

  • Primary recommendation: TE buffer at pH 9.0

  • Alternative approach: Citrate buffer at pH 6.0

• Protocol details:

  • For FFPE (formalin-fixed paraffin-embedded) sections: deparaffinize and rehydrate through graded alcohols to water

  • Immerse slides in retrieval buffer in a suitable container

  • Heat using pressure cooker, microwave, or water bath to 95-100°C

  • Maintain at temperature for 15-20 minutes

  • Allow to cool to room temperature (approximately 20 minutes)

  • Rinse thoroughly in PBS or TBS before proceeding with immunostaining

• Tissue-specific considerations:

  • Human heart and pancreas tissues have been verified to show positive IHC results with these retrieval methods

  • Mouse lung tissue has also demonstrated successful staining

• When using HRP-conjugated antibodies:

  • Include additional peroxidase blocking step (3% H₂O₂ in methanol for 10 minutes)

  • Perform protein blocking with 5% normal serum or commercial blocking reagents

  • Optimize antibody dilution (typically 1:50-1:500 range)

These antigen retrieval methods have been empirically determined to provide optimal FBLN5 detection while maintaining tissue morphology and minimizing background staining. Researchers should validate and potentially modify these protocols based on their specific tissue samples and experimental conditions.

How should sample preparation be optimized for detecting FBLN5 in different tissue types?

Optimal detection of FBLN5 requires tissue-specific sample preparation approaches due to its varied expression and localization patterns across different tissues. The following guidelines can help researchers maximize detection sensitivity and specificity:

• Tissue-specific lysis considerations:

  • Heart tissue: Use RIPA buffer supplemented with protease inhibitors; brief sonication improves extraction

  • Brain tissue: Consider using specialized neuronal tissue lysis buffers that maintain protein integrity

  • Vascular samples: Include detergents that effectively solubilize membrane-associated proteins

  • Placenta and kidney: May require more stringent extraction conditions due to high protease content

• Protein extraction optimization:

  • For Western blot: Include 1-5 mM EDTA to preserve calcium-binding EGF domains

  • For immunoprecipitation: Extraction in non-denaturing conditions preserves protein-protein interactions

  • For immunohistochemistry: Fixation in 4% paraformaldehyde for 24 hours provides optimal results

• Sample processing by application:

  Application	Sample Processing Recommendation
  WB	For heart tissue: 1:1000-1:8000 dilution yields optimal results
  IP	0.5-4.0 μg antibody for 1.0-3.0 mg total protein
  IHC	1:50-1:500 dilution with tissue-specific optimization

• Tissue-specific positive controls:

  • Mouse heart, brain, placenta, skeletal muscle, and kidney tissues

  • Rat brain and heart tissues

  • Human heart and pancreas tissues

• Special considerations for glycosylated forms:

  • Different tissues may show varying glycosylation patterns

  • Heart tissue typically shows the 65 kDa fully glycosylated form

  • Brain tissue may show multiple bands representing various glycosylation states

By tailoring sample preparation methods to tissue-specific characteristics, researchers can enhance the detection of FBLN5 while minimizing artifacts and optimizing signal-to-noise ratios in their experimental systems.

What blocking conditions are optimal for reducing background when using HRP-conjugated FBLN5 antibodies?

Minimizing background signal is particularly important when using HRP-conjugated antibodies due to their direct enzyme activity. For FBLN5 HRP-conjugated antibodies, the following blocking conditions have been empirically determined to provide optimal signal-to-noise ratios:

• Recommended blocking buffers:

  • Western blotting: 5% non-fat dry milk (NFDM) in TBST has shown excellent results

  • Immunohistochemistry: 5-10% normal serum (species matched to secondary antibody) in PBS with 0.1-0.3% Triton X-100

  • ELISA: 1-5% BSA in PBS with 0.05% Tween-20

• Blocking protocol optimization:

  • Duration: 1 hour at room temperature or overnight at 4°C

  • Temperature effect: Room temperature blocking is generally sufficient, but background-prone tissues may benefit from 4°C overnight blocking

  • Buffer additives: Addition of 0.1% Tween-20 can further reduce non-specific binding

• Endogenous peroxidase quenching (critical for HRP-conjugated antibodies):

  • Treat samples with 0.3-3% H₂O₂ in methanol for 10-30 minutes prior to blocking

  • For highly vascularized tissues (which may express FBLN5), increase H₂O₂ concentration to 3%

  • Allow complete drying after peroxidase quenching before applying blocking solution

• Tissue-specific considerations:

  • Heart tissue: May require additional blocking with avidin/biotin blocking kit due to endogenous biotin

  • Lung tissue: Add 0.1% fish gelatin to blocking buffer to reduce background

  • Skin samples: Include 0.1-0.3% Triton X-100 to improve antibody penetration

• Antibody diluent composition:

  • Dilute antibodies in the same blocking buffer at reduced concentration (typically 1-10% of blocking concentration)

  • For Western blotting, validated dilution ranges of 1:1000-1:8000 in 5% NFDM/TBST yield optimal results

These optimized blocking conditions help ensure specific detection of FBLN5 while minimizing background interference, particularly important when investigating tissues with complex extracellular matrix composition where non-specific binding can be problematic.

What are common issues when using FBLN5 antibodies and how can they be resolved?

Researchers working with FBLN5 antibodies may encounter several technical challenges. The following troubleshooting guide addresses common issues and provides evidence-based solutions:

• Multiple bands in Western blot:

  • Issue: Detection of bands at 50 kDa, 55 kDa, and 65-66 kDa

  • Explanation: Different glycosylation states; 66 kDa corresponds to fully glycosylated protein

  • Solution: Use glycosidase treatment to confirm glycosylation status; run longer separation gels for better resolution; include positive control tissues known to express specific forms

• Weak or no signal:

  • Potential causes: Insufficient antigen retrieval; antibody concentration too low; protein degradation

  • Solutions:

    • For IHC: Optimize antigen retrieval using recommended TE buffer pH 9.0 or citrate buffer pH 6.0

    • For WB: Decrease antibody dilution (start with 1:1000 rather than 1:8000)

    • Add protease/phosphatase inhibitors to prevent degradation

    • Verify positive control tissues (mouse heart, brain samples are reliable positive controls)

• High background signal:

  • Solutions:

    • Increase blocking time/concentration (5% NFDM/TBST recommended)

    • For HRP-conjugated antibodies, ensure thorough peroxidase quenching

    • Increase washing steps (at least 3×10 minutes with TBST)

    • Optimize antibody dilution based on signal-to-noise ratio

• Inconsistent results across tissue types:

  • Explanation: Tissue-specific expression and glycosylation patterns

  • Solution: Adjust protein loading (10-20 μg recommended for most tissues); optimize extraction protocols for specific tissues; consider tissue-specific positive controls

• Non-specific bands:

  • Solutions:

    • Perform antibody validation using FBLN5 knockdown/knockout controls

    • Increase antibody specificity by using monoclonal antibodies

    • Optimize washing conditions (increase detergent concentration slightly)

By implementing these troubleshooting approaches, researchers can overcome common technical challenges associated with FBLN5 antibody applications and generate more reliable, reproducible results across different experimental systems.

How can FBLN5 antibodies be used to investigate hypoxia-related vascular pathologies?

FBLN5 antibodies provide valuable tools for investigating hypoxia-related vascular pathologies, given the established relationship between hypoxia and FBLN5 regulation. Strategic experimental approaches include:

• Monitoring hypoxia-induced changes in FBLN5 expression:

  • Western blot analysis reveals approximately 2.5-fold increase in FBLN5 protein levels after 24 hours of hypoxia exposure

  • Use HRP-conjugated antibodies at 1:1000-1:8000 dilution for optimal detection of both intracellular and extracellular FBLN5 protein levels

  • Compare normoxic vs. hypoxic conditions across different timepoints (maximal induction at 24h)

• Investigating signaling pathways:

  • Combine FBLN5 antibody detection with inhibitors of PI3K/Akt/mTOR pathway (e.g., LY294002, rapamycin)

  • Use alongside HIF-1α detection methods to correlate HIF-1α stabilization with FBLN5 upregulation

  • Pair with dimethyl oxalylglycine treatment to verify HIF-1α involvement

• Correlation with pathological vascular remodeling:

  • IHC analysis (1:50-1:500 dilution) of FBLN5 in atherosclerotic plaques and areas of neointimal thickening

  • Dual immunofluorescence with endothelial/smooth muscle markers to identify cell-specific expression

  • Quantitative analysis of FBLN5 distribution in vascular lesions

• Functional studies:

  • Use FBLN5 antibodies to neutralize protein function in experimental models

  • Combine with apoptosis assays to evaluate FBLN5's protective role during hypoxic stress

  • Correlate with matrix assembly markers to assess ECM remodeling under hypoxic conditions

• Translational applications:

  • Analysis of FBLN5 expression in patient-derived vascular samples from hypoxia-related pathologies

  • Correlation with disease progression and severity markers

  • Potential development of diagnostic applications based on FBLN5 expression patterns

These approaches leverage the specificity of FBLN5 antibodies to investigate the molecular mechanisms linking hypoxia, FBLN5 regulation, and vascular pathophysiology, potentially identifying new therapeutic targets for vascular diseases associated with hypoxic conditions.

What are the considerations for using FBLN5 antibodies in multiplex immunostaining protocols?

Incorporating FBLN5 antibodies into multiplex immunostaining protocols requires careful consideration of several technical factors to ensure successful co-detection with other markers. The following guidelines will help researchers optimize multiplex approaches:

• Antibody compatibility assessment:

  • Species origin considerations: FBLN5 antibodies are typically rabbit-derived; pair with mouse, goat, or rat antibodies for other markers to avoid cross-reactivity

  • Isotype compatibility: When using multiple rabbit antibodies, consider sequential detection methods or directly conjugated primary antibodies

• Optimization of multiplex panel:

  Panel Component	Recommendation for FBLN5 Multiplex
  FBLN5 detection	1:50-1:500 dilution, TE buffer pH 9.0 antigen retrieval
  ECM markers	Collagen, elastin, fibrillin-1 (compatible with FBLN5 detection)
  Cell-type markers	CD31 (endothelial), α-SMA (smooth muscle), CD68 (macrophages)
  Hypoxia markers	HIF-1α, CA9, GLUT1 (for hypoxia studies)

• Sequential vs. simultaneous staining protocols:

  • Sequential approach: Recommended when antibodies require different antigen retrieval methods

  • Simultaneous approach: Suitable when antibodies share compatible conditions

  • For HRP-conjugated antibodies: Sequential detection using tyramide signal amplification allows multiplexing

• Cross-reactivity mitigation strategies:

  • Include blocking steps between detection sequences

  • Use highly cross-adsorbed secondary antibodies

  • Implement spectral unmixing for fluorescent detection systems

• Validation of multiplex results:

  • Compare multiplex staining patterns with single-marker controls

  • Include appropriate blocking controls for each antibody in the panel

  • Verify signal specificity using FBLN5 knockdown controls

• Tissue-specific considerations:

  • Vascular tissues: Special attention to autofluorescence quenching

  • Lung tissue: Additional blocking steps may be required

  • Heart tissue: Shows strong FBLN5 expression; may require antibody titration

By carefully addressing these considerations, researchers can successfully integrate FBLN5 detection into multiplex immunostaining protocols, enabling simultaneous visualization of FBLN5 alongside other markers of interest for comprehensive analysis of tissue microenvironments in health and disease.

How can FBLN5 antibodies be used to investigate the role of FBLN5 in age-related pathologies?

FBLN5 has been implicated in several age-related pathologies, including age-related macular degeneration and vascular aging. FBLN5 antibodies offer valuable tools for investigating these connections through various experimental approaches:

• Analysis of FBLN5 expression changes during aging:

  • Western blot analysis (1:1000-1:8000 dilution) to quantify age-dependent changes in FBLN5 expression across tissues

  • IHC (1:50-1:500 dilution) to visualize alterations in FBLN5 distribution and localization in aged tissues

  • Comparison of glycosylation patterns (50-65 kDa bands) between young and aged tissues

• Investigation of FBLN5 mutations in age-related disorders:

  • Use of antibodies that specifically recognize wild-type but not mutant FBLN5

  • Analysis of FBLN5 protein levels in patient samples with age-related macular degeneration

  • Correlation of FBLN5 expression with disease progression

• Assessment of elastin organization and integrity:

  • Co-immunostaining of FBLN5 with elastin and other elastic fiber components

  • Quantitative analysis of FBLN5-elastin colocalization in young versus aged tissues

  • Correlation with functional measures of tissue elasticity

• FBLN5 in vascular aging studies:

  • Analysis of FBLN5 distribution in aged vessels with atherosclerotic plaques

  • Correlation with markers of vascular stiffening and endothelial dysfunction

  • Investigation of hypoxia-related changes in FBLN5 expression in aged vessels

• Mechanistic studies of FBLN5 in cellular senescence:

  • Detection of FBLN5 in senescent cell secretome

  • Analysis of FBLN5-integrin interactions in aged cells

  • Investigation of FBLN5's role in ECM remodeling during aging

These approaches leverage the specificity of FBLN5 antibodies to dissect the complex roles of this protein in age-related pathologies, potentially identifying new therapeutic targets and biomarkers for age-associated diseases. The combined use of different techniques (WB, IHC, IP) provides complementary insights into FBLN5's changing expression, localization, and function during aging processes.