FBL25 Antibody

What criteria should researchers consider when selecting an appropriate FBLN5 antibody for their experiments?

When selecting a FBLN5 antibody, researchers should evaluate several critical parameters:

• Target species reactivity: Confirm antibody reactivity with your experimental species (human, mouse, rat)

• Antibody type: Consider whether monoclonal or polyclonal is more suitable for your application

• Validated applications: Verify the antibody has been validated for your specific application (WB, IHC, IF)

• Epitope/immunogen: Review the antibody's target region on FBLN5 to ensure it's accessible in your experimental conditions

• Molecular weight recognition: Check if the antibody detects the expected molecular weight (typically 65-70 kDa for FBLN5)

Most commercial FBLN5 antibodies have documented reactivity across human, mouse, and rat samples, but validation data varies significantly between vendors. For instance, Proteintech's 29992-1-AP FBLN5 antibody has been validated in multiple tissues including mouse heart, mouse kidney, and rat heart for Western blotting applications .

How can I verify the specificity of an FBLN5 antibody before conducting my main experiments?

Basic methodological approach:

Antibody specificity verification requires multiple control experiments:

• Positive control tissues: Use tissues known to express FBLN5, such as heart, lung, and vascular tissues

• Western blot validation: Confirm the antibody detects the expected 65-70 kDa band in positive control samples

• Negative control tissues/cells: Test tissues/cells with minimal FBLN5 expression

• Control antibodies: Include isotype controls to assess non-specific binding

• Knockout/knockdown validation: If available, test the antibody against FBLN5 knockout or knockdown samples

For optimal verification, compare results across multiple detection methods. As shown in validation data, FBLN5 antibodies typically show strong signals in heart, lung, and vascular tissues, with observed molecular weights between 65-70 kDa despite a calculated molecular weight of approximately 50 kDa .

What are the optimal sample preparation methods for detecting FBLN5 in different experimental contexts?

Sample preparation methods vary by application and tissue type:

Western Blot sample preparation:

• Extract proteins using RIPA buffer with protease inhibitors

• Include reducing agent (β-mercaptoethanol) in loading buffer

• Heat samples at 95°C for 5 minutes before loading

• Load 20-50 μg of protein per lane for optimal detection

Immunohistochemistry preparation:

• Fix tissues in 10% neutral buffered formalin

• For paraffin sections, perform antigen retrieval using TE buffer pH 9.0

• Alternatively, use citrate buffer pH 6.0 for antigen retrieval

• Block with 5-10% serum from the same species as secondary antibody

Recommended dilutions by application:

Sample-dependent optimization is essential, as FBLN5 expression levels vary significantly between tissues and disease states .

What are common issues encountered when detecting FBLN5 in experimental samples, and how can they be resolved?

Advanced research question:

Researchers frequently encounter several technical challenges when working with FBLN5 antibodies:

• High background in IHC/IF:

  • Increase blocking time (2 hours at room temperature)

  • Use 0.1% sodium azide in washing buffers to prevent internalization of membrane antigens

  • Perform all protocol steps on ice to prevent antigen internalization

  • Ensure cell viability >90% as dead cells show high background scatter

• Multiple bands in Western blot:

  • FBLN5 undergoes post-translational modifications, resulting in observed molecular weight (65-70 kDa) higher than calculated (50 kDa)

  • Verify band specificity with knockout/knockdown controls

  • Test multiple antibodies targeting different epitopes

• Weak or no signal:

  • For secreted proteins like FBLN5, concentrate conditioned media using TCA precipitation

  • Check cell culture conditions that might affect FBLN5 expression

  • Verify tissue-specific expression patterns (FBLN5 is highly expressed in vascular tissues, lungs, and heart)

• Inconsistent results between applications:

  • Different epitopes may be accessible in different applications

  • Some antibodies perform better in native conditions (IF/IHC) than denaturing conditions (WB)

How should dilution series be designed to optimize FBLN5 antibody performance in various applications?

Methodological approach:

Systematic antibody dilution optimization requires:

• Initial broad-range titration:

  • For Western blot: Test 1:500, 1:2000, 1:5000, and 1:10000 dilutions

  • For IHC: Test 1:50, 1:100, 1:200, and 1:500 dilutions

• Secondary antibody optimization:

  • Match secondary antibody host species to primary antibody

  • Test secondary antibody at multiple dilutions (typically 1:1000 to 1:10000)

  • Include secondary-only controls to assess non-specific binding

• Exposure time/development optimization:

  • For WB: Test multiple exposure times

  • For IHC/IF: Optimize development time with chromogenic substrates

• Sample-specific adjustments:

  • Different tissues may require different antibody concentrations

  • Disease states may alter FBLN5 expression levels requiring adjusted protocols

Remember that antibody titration should be performed for each new tissue type and experimental condition to ensure optimal signal-to-noise ratios.

How can FBLN5 antibodies be effectively used in co-immunoprecipitation studies to investigate protein-protein interactions?

Advanced methodological approach:

FBLN5 interacts with multiple proteins including tropoelastin, fibrillin-1, superoxide dismutase (SOD3), and various integrins . Co-immunoprecipitation (Co-IP) requires:

• Sample preparation:

  • Use gentle lysis buffers (e.g., 25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 5% glycerol)

  • Include protease inhibitors to prevent degradation

  • Perform all steps at 4°C

• Pre-clearing:

  • Pre-clear lysates with protein A/G beads to reduce non-specific binding

  • Pre-incubate antibody with beads before adding lysate

• Antibody selection:

  • Use monoclonal antibodies for higher specificity in Co-IP

  • Consider epitope location - antibodies targeting functional domains may disrupt protein interactions

• Controls:

  • Include isotype control antibodies

  • Use lysates from FBLN5 knockdown cells as negative controls

• Elution and detection:

  • Use gentle elution conditions to preserve protein-protein interactions

  • Detect interacting partners via Western blot or mass spectrometry

Known FBLN5 protein interactions that can be investigated:

What considerations are important when using FBLN5 antibodies in different cellular fractionation studies?

Advanced research question:

FBLN5 is primarily a secreted extracellular protein but may also be found in various cellular compartments during synthesis and processing. Key considerations include:

• Compartment-specific isolation:

  • Use differential centrifugation to separate cellular fractions

  • For secreted FBLN5: Collect and concentrate conditioned media

  • For intracellular FBLN5: Separate ER/Golgi fractions where protein synthesis occurs

• Marker proteins for fraction validation:

  • Include controls for each cellular compartment (e.g., calnexin for ER, GM130 for Golgi)

  • Verify fraction purity before analyzing FBLN5 distribution

• Sample processing:

  • Process samples immediately to prevent degradation

  • Use different detergents based on target compartment (e.g., digitonin for cytosolic proteins, Triton X-100 for membrane proteins)

• Antibody selection:

  • Use antibodies recognizing different epitopes as processing may alter accessibility

  • Consider native vs. denatured conditions based on compartment

• Quantification methods:

  • Use densitometry to compare FBLN5 levels across fractions

  • Normalize to loading controls specific to each cellular compartment

Research shows that FBLN5 typically follows the secretory pathway through the ER and Golgi before being secreted into the extracellular space, where it associates with elastic fibers and interacts with cell surface receptors .

How can FBLN5 antibodies be applied to investigate elastic fiber abnormalities in disease models?

Advanced methodological approach:

FBLN5 plays a critical role in elastic fiber formation, and its dysregulation is implicated in several diseases:

• Disease model selection:

  • Fbln5 knockout mice exhibit systemic elastic fiber defects including loose skin, tortuous aorta, emphysematous lung, and genital prolapse

  • Human conditions associated with FBLN5 mutations include age-related macular degeneration and cutis laxa

• Tissue-specific analysis:

  • Vascular tissues: Analyze aortic wall structure and elasticity

  • Pulmonary tissues: Examine alveolar structure and elastin organization

  • Skin samples: Assess dermal elastic fiber network

• Multimodal analysis:

  • Combine immunostaining with histological elastic fiber stains (Verhoeff-Van Gieson)

  • Use electron microscopy to examine ultrastructural abnormalities

  • Perform mechanical testing to correlate structural changes with functional outcomes

• Quantitative assessment:

  • Measure elastic fiber length, thickness, and organization

  • Analyze elastic fiber density in affected tissues

  • Correlate FBLN5 expression levels with elastic fiber abnormalities

Research shows that Fbln5-null tissues contain only short, disrupted elastic fibers without affecting collagen fiber formation, confirming that defects are specific to elastic fiber assembly . These findings establish valuable models for studying congenital elastic fiber disorders.

What protocols should be followed when using FBLN5 antibodies to evaluate therapeutic interventions targeting elastic tissue regeneration?

Advanced research question:

Evaluating therapeutic interventions for elastic tissue regeneration using FBLN5 antibodies requires:

• Baseline assessment:

  • Quantify FBLN5 expression and localization in target tissues before intervention

  • Characterize elastic fiber architecture using immunohistochemistry and specialized stains

  • Document mechanical properties of elastic tissues

• Intervention monitoring:

  • Track changes in FBLN5 expression during therapy using quantitative methods (qPCR, Western blot)

  • Assess FBLN5 localization to newly forming elastic fibers via co-localization studies

  • Monitor elastic fiber maturation through temporal sampling

• Multiple antibody approach:

  • Use antibodies against different elastic fiber components (FBLN5, elastin, fibrillin-1)

  • Employ antibodies recognizing different FBLN5 epitopes to ensure comprehensive detection

• Functional correlation:

  • Correlate FBLN5 expression with mechanical testing results

  • Assess elastic fiber functionality using physiological measurements

  • Document clinical improvements in relation to FBLN5/elastic fiber restoration

Given that FBLN5 regulates coacervation efficiency and controls the size of elastin self-aggregates during elastic fiber assembly , monitoring these parameters can provide valuable insights into therapeutic effectiveness for conditions such as cutis laxa, emphysema, and vascular elastinopathies.