FABP3 Paired Antibody

What is FABP3 and what biological roles does it play in cellular function?

FABP3 is a small 15-kDa cytoplasmic protein primarily expressed in heart and skeletal muscle tissue. It functions as a carrier protein for polyunsaturated fatty acids (PUFAs) and participates in multiple cellular functions . FABP3 transports fatty acids and other lipophilic substances from the cytoplasm to the nucleus, playing critical roles in:

• Intracellular fatty acid transport and metabolism

• Regulation of gene expression through delivery of lipid signaling molecules to nuclear receptors

• Modulation of inflammatory responses

• B-cell activation through histone acetylation control

• Plasma cell development and immunoglobulin M (IgM) secretion

Research has demonstrated that FABP3 is necessary for Blimp-1 expression, functioning as a positive regulator of B-cell activation by controlling histone acetylation of the Blimp-1 gene, thereby contributing to host defense against pathogens .

What specific applications are FABP3 Paired Antibodies validated for?

FABP3 Paired Antibodies are validated for multiple research applications as shown in the following table:

When designing experiments, researchers should note that optimal dilutions are sample-dependent and may require titration in each specific testing system to achieve optimal results .

How should FABP3 Paired Antibodies be stored and handled for maximum stability?

For optimal stability and performance of FABP3 Paired Antibodies:

• Store at 4°C if the entire vial will be used within 2-4 weeks

• For longer periods, store frozen at -20°C

• Antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Aliquoting is unnecessary for -20°C storage

• Some formulations contain 0.1% BSA in small sizes (20μl)

• Allow antibodies to reach room temperature before opening vials

• Avoid repeated freeze-thaw cycles to maintain antibody integrity

• When developing immunoassays, both capture and detection antibodies should be handled according to their specific storage requirements

Proper storage and handling maintain the structural integrity and binding capacity of the antibodies, ensuring consistent experimental results.

What are the critical validation steps for confirming FABP3 antibody specificity in different tissue types?

When validating FABP3 antibodies for new tissue types or experimental systems, researchers should implement the following validation steps:

• Cross-reactivity testing: Validate reactivity across species by testing on human, mouse, and rat samples. Current FABP3 antibodies show tested reactivity with human, mouse, and rat samples .

• Multiple detection methods: Confirm specificity using at least two independent techniques (e.g., Western blot plus immunohistochemistry).

• Molecular weight verification: Confirm that the observed molecular weight matches the calculated weight of 15 kDa for FABP3 .

• Positive and negative controls: Include appropriate tissue controls:

  • Positive controls: Human, mouse, or rat heart tissue (high FABP3 expression)

  • Negative controls: Tissues known to have minimal FABP3 expression or knockout/knockdown samples

• Expression pattern analysis: Verify that the observed tissue distribution matches known FABP3 expression patterns (primarily in heart and skeletal muscle, but with skeletal muscle concentration approximately 20 times lower than cardiac tissue) .

• Blocking peptide competition: Use specific FABP3 blocking peptides to confirm signal specificity.

This systematic validation approach ensures antibody specificity before proceeding with complex experimental designs or clinical studies.

How can researchers optimize FABP3 detection in multiplex immunoassays?

Optimizing FABP3 detection in multiplex immunoassays requires careful consideration of several parameters:

• Antibody pair selection: Choose validated capture and detection antibody pairs that don't interfere with other targets in your multiplex panel. The specific clones in FABP3 Paired Antibody kits have been validated for compatibility .

• Cross-reactivity mitigation: Test for potential cross-reactivity with other FABPs, particularly FABP4 and FABP5, which are structurally similar and may be co-expressed in certain tissues .

• Buffer optimization: Adjust assay buffers to minimize background while maintaining specific signal:

  • For capture antibody coating: Standard carbonate-bicarbonate buffer (pH 9.6)

  • For sample dilution: PBS with 0.05% Tween-20 and 1-2% BSA to reduce non-specific binding

• Signal enhancement: When detecting low abundance FABP3, employ signal amplification methods such as:

  • Biotin-streptavidin systems

  • Polymer-based detection systems

  • Tyramide signal amplification

• Calibration curve design: Prepare a multi-point calibration curve using recombinant FABP3 standards to ensure accurate quantification across the physiological range (typically 0.5-20 ng/mL for cardiac applications).

• Assay validation metrics: Establish and document key performance indicators:

  • Limit of detection (typically sub-ng/mL)

  • Precision (%CV < 15%)

  • Accuracy (80-120% recovery)

  • Linearity across the measurement range

Implementing these optimization strategies will maximize sensitivity and specificity when measuring FABP3 alongside other biomarkers in complex biological samples.

How do FABP3 interactions with other proteins influence cellular signaling pathways?

Research using multi-modal imaging approaches has revealed that FABP3 forms complex higher-order assemblies with other proteins involved in cellular signaling, particularly in eicosanoid biosynthesis . These interactions have significant implications for understanding inflammatory responses and lipid signaling.

Key FABP3 protein interactions and their signaling effects include:

• Interactions with eicosanoid biosynthetic enzymes:

  • FABP3 interacts with 5-lipoxygenase activating protein (FLAP)

  • FABP3 forms complexes with cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2)

  • These interactions influence prostaglandin and leukotriene synthesis

• Response to inflammatory stimuli:

  • After lipopolysaccharide (LPS) stimulation, the percentage of COX-1 associated with FABP3 increases from 17% to 21%

  • Similarly, COX-2 association with FABP3 increases from 17% to 31% following LPS stimulation

• Comparison with other FABPs:

  • FABP3, FABP4, and FABP5 are expressed in macrophages and form different patterns of association with inflammatory enzymes

  • These distinct interaction patterns suggest specialized roles in inflammatory signaling

• Nuclear translocation and gene regulation:

  • FABP3 can translocate to the nucleus and modulate gene expression

  • In B cells, FABP3 regulates Blimp-1 expression through histone acetylation

Understanding these protein-protein interactions provides insight into how FABP3 contributes to cellular signaling networks, particularly in inflammatory responses and immune cell activation.

What role does FABP3 play in B-cell differentiation and antibody production?

Recent research has established FABP3 as a critical regulator in B-cell biology, particularly in the differentiation of antibody-producing plasma cells . The mechanisms through which FABP3 influences B-cell function include:

• Epigenetic regulation:

  • FABP3 controls histone acetylation of the Blimp-1 gene promoter region

  • This epigenetic modification is essential for Blimp-1 expression, a master regulator of plasma cell development

• Plasma cell differentiation:

  • FABP3 promotes the development of plasma cells from activated B cells

  • This process is crucial for efficient humoral immune responses to pathogens

• IgM production:

  • FABP3 specifically enhances IgM secretion from plasma cells

  • This indicates a role in early antibody responses before class switching occurs

• Metabolic regulation:

  • As a fatty acid transporter, FABP3 likely influences B-cell metabolism

  • Fatty acid metabolism is increasingly recognized as important for immune cell activation and function

These findings establish FABP3 as a positive regulator of B-cell activation with direct implications for humoral immunity and host defense against pathogens. Researchers studying B-cell biology should consider FABP3 as a potential target for modulating antibody responses in various disease contexts.

How can FABP3 detection be integrated into multiparameter flow cytometry protocols?

Integrating FABP3 detection into multiparameter flow cytometry requires careful optimization due to its primarily intracellular localization. Here is a methodological approach for researchers:

• Sample preparation and fixation:

  • Fix cells with 2-4% paraformaldehyde for 10-15 minutes at room temperature

  • Permeabilize with 0.1-0.5% saponin or 0.1% Triton X-100 to allow antibody access to intracellular FABP3

• Antibody selection and titration:

  • Use monoclonal anti-FABP3 antibodies (such as Mouse IgG2a monoclonal)

  • Perform antibody titration (typically starting at 1:200-1:800 dilution for flow cytometry)

  • Select fluorophores that complement other markers in your panel (consider spectral overlap)

• Multiparameter panel design:

  • For immune cell studies, combine with surface markers (CD19, CD138 for B cells/plasma cells)

  • For cardiac/muscle research, combine with markers like cardiac troponin or α-actinin

  • Include markers for cell viability and activation status

• Controls and validation:

  • Use FMO (fluorescence minus one) controls to set proper gates

  • Include positive controls (heart/muscle cell lines with known FABP3 expression)

  • Validate findings with Western blot or immunofluorescence microscopy

• Data analysis considerations:

  • Analyze FABP3 expression as mean fluorescence intensity (MFI)

  • Consider using dimensionality reduction methods (tSNE, UMAP) for complex datasets

  • Correlate FABP3 expression with functional parameters in your experimental system

This methodological approach enables researchers to study FABP3 expression at the single-cell level and correlate it with other cellular parameters in heterogeneous populations.

What are common pitfalls in FABP3 immunoassay development and how can they be addressed?

Researchers developing FABP3 immunoassays frequently encounter several technical challenges. Here are the most common issues and their solutions:

• Cross-reactivity with other FABP family members:

  • Problem: FABP3, FABP4, and FABP5 share structural similarities and may cause cross-reactivity

  • Solution: Use monoclonal antibodies specifically validated against multiple FABP family members

  • Validation approach: Test antibody specificity against recombinant FABP1-7 proteins

• Homodimer detection:

  • Problem: Western blots sometimes show unexpected bands at approximately twice the expected size (~30 kDa) due to FABP homodimer formation

  • Solution: Include reducing agents (DTT or β-mercaptoethanol) in sample preparation and heat samples adequately before electrophoresis

• Tissue-specific optimization requirements:

  • Problem: Different tissues require different antigen retrieval and detection protocols

  • Solution: For heart tissue, use TE buffer pH 9.0 for antigen retrieval; alternatively, citrate buffer pH 6.0 may be used for other tissues

• Sample matrix effects in plasma/serum testing:

  • Problem: Plasma components can interfere with antibody binding

  • Solution: Optimize sample dilution (typically 1:2 to 1:10) and use specialized assay buffers containing blocking agents

• Hook effect in high-concentration samples:

  • Problem: Very high FABP3 concentrations (as in acute cardiac injury) can cause false negative or low results

  • Solution: Test samples at multiple dilutions when high FABP3 levels are suspected

• Stability of detection antibody conjugates:

  • Problem: Gold-conjugated detection antibodies may aggregate over time

  • Solution: Store according to manufacturer recommendations and centrifuge before use to remove any aggregates

Addressing these common pitfalls enables researchers to develop robust and reliable FABP3 immunoassays for both research and clinical applications.

How can researchers distinguish between FABP3 and other FABP family members in complex tissue samples?

Distinguishing between closely related FABP family members is a significant challenge in complex tissues where multiple FABPs may be co-expressed. Researchers can implement the following methodological approaches to achieve specific FABP3 detection:

• Selection of highly specific antibodies:

  • Choose monoclonal antibodies raised against unique epitopes of FABP3

  • Verify specificity against recombinant FABP family members (particularly FABP1, FABP4, and FABP5)

  • The Mouse IgG2a monoclonal antibody targeting specific FABP3 epitopes shows high specificity

• Differential expression analysis:

  • Leverage tissue-specific expression patterns: FABP3 is predominantly expressed in heart and skeletal muscle

  • FABP3 concentration is approximately 20 times higher in cardiac tissue than in skeletal muscle, unlike myoglobin which has similar concentrations in both tissues

• Multi-antibody approach:

  • Use multiple antibodies targeting different FABP3 epitopes

  • Confirm specificity through co-localization studies

• Mass spectrometry validation:

  • Employ targeted proteomics to identify specific FABP3 peptides that differ from other FABP family members

  • This approach can definitively distinguish between highly homologous proteins

• Genetic approaches:

  • Use siRNA/shRNA knockdown of FABP3 to confirm antibody specificity

  • For animal studies, consider FABP3 knockout models as negative controls

• Combined immunoprecipitation and Western blotting:

  • Immunoprecipitate with anti-FABP3 antibody and confirm by Western blot

  • This two-step approach increases specificity for FABP3 detection

These methodological strategies enable researchers to accurately detect and quantify FABP3 in complex biological samples containing multiple FABP family members.

What are the latest developments in FABP3's role in inflammatory signaling and eicosanoid biosynthesis?

Recent advanced imaging studies have revealed novel insights into FABP3's involvement in inflammatory signaling pathways, particularly in relation to eicosanoid biosynthesis . These findings represent significant developments in understanding FABP3's broader physiological roles:

• Formation of higher-order protein assemblies:

  • Multi-modal imaging combining direct stochastic optical reconstruction microscopy (dSTORM) with computational analyses and fluorescence lifetime imaging microscopy (FLIM) has demonstrated that FABP3 forms dynamic protein clusters with key enzymes in eicosanoid biosynthesis

  • These higher-order assemblies are thought to facilitate efficient signaling by creating specialized microenvironments within cells

• Dynamic interactions with COX enzymes:

  • FABP3 shows differential interactions with cyclooxygenase isoforms:

    • FABP3-COX-1 interactions: 17% colocalization before LPS stimulation, increasing to 21% after stimulation

    • FABP3-COX-2 interactions: 17% colocalization before LPS stimulation, significantly increasing to 31% after stimulation

  • These findings suggest that FABP3 preferentially interacts with COX-2 during inflammatory responses

• FLAP interactions in eicosanoid synthesis:

  • FABP3 also forms complexes with 5-lipoxygenase activating protein (FLAP)

  • These interactions may influence the synthesis of leukotrienes in addition to prostaglandins

• Quality vs. quantity of protein interactions:

  • Fluorescence lifetime imaging reveals that the quality of interaction (measured by changes in fluorescence lifetime) between FABP3 and inflammatory enzymes may be more critical than the quantity of interacting molecules

  • This suggests that conformational changes in these protein complexes are important for signaling

These discoveries provide a molecular framework for understanding how FABP3 contributes to inflammatory responses, beyond its classical role as a fatty acid transporter.

How can FABP3 be utilized as a cardiac biomarker in multiplex diagnostic platforms?

FABP3 (heart-type fatty acid-binding protein) has emerged as a valuable cardiac biomarker with distinct advantages for multiplex diagnostic platforms. Its rapid release kinetics following cardiac injury make it particularly valuable for early detection of myocardial damage :

• Early detection capabilities:

  • FABP3 appears in plasma within 2 hours after acute myocardial infarction (AMI)

  • Returns to normal levels within 18-24 hours

  • This rapid kinetic profile makes it ideal for early assessment of cardiac injury

• Advantages over traditional markers:

  • Compared to myoglobin (another early marker):

    • FABP3 concentration is 20 times lower in skeletal muscle than in cardiac tissue

    • This makes FABP3 more cardiac-specific than myoglobin, which has equivalent concentrations in cardiac and skeletal tissue

  • Compared to troponins:

    • Earlier rise (1-3 hours vs. 4-6 hours for troponins)

    • Complements the high specificity of troponins with earlier detection capability

• Integration into multiplex platforms:

  • FABP3 Paired Antibody kits enable lateral flow immunoassay development

  • Optimized antibody pairs (capture and detection) facilitate integration into multimarker panels

  • Compatible with various detection methods (colorimetric, fluorescent, electrochemical)

• Clinical applications in multiplex settings:

  • Early rule-out of AMI in chest pain patients

  • Estimation of myocardial infarct size

  • Monitoring reperfusion in STEMI patients

  • Detection of perioperative myocardial injury

• Methodological considerations for multiplex integration:

  • Optimal antibody dilutions: capture antibody at ~2 μg/mL and detector antibody at ~0.5 μg/mL

  • Sample preparation: plasma or serum samples with minimal preprocessing

  • Detection window: focus on 2-12 hours after symptom onset for maximum clinical utility

When properly implemented in multiplex diagnostic platforms, FABP3 provides valuable complementary information to traditional cardiac markers, enabling more accurate and timely diagnosis of cardiac injury.

What are promising areas for future research on FABP3's role in cellular metabolism and signaling?

Based on current understanding of FABP3 biology, several promising research directions emerge for investigating its broader roles in cellular metabolism and signaling:

• Integration of lipid metabolism and immune function:

  • Investigate how FABP3-mediated fatty acid transport influences metabolic reprogramming during immune cell activation

  • Explore connections between FABP3, fatty acid oxidation, and B-cell differentiation

  • Study the role of FABP3 in delivering specific fatty acids to nuclear receptors for transcriptional regulation

• FABP3 in extracellular vesicle biology:

  • Examine whether FABP3 is packaged into extracellular vesicles (EVs)

  • Investigate potential paracrine signaling roles of FABP3-containing EVs

  • Explore EV-FABP3 as a biomarker for tissue-specific damage

• Post-translational modifications of FABP3:

  • Characterize how phosphorylation, acetylation, or other modifications alter FABP3 function

  • Investigate whether these modifications regulate FABP3's interaction with binding partners or subcellular localization

  • Develop antibodies specific to modified FABP3 for studying these processes

• FABP3 in cellular stress responses:

  • Explore FABP3's role in oxidative stress and hypoxia responses

  • Investigate connections between FABP3 and ER stress pathways

  • Study how FABP3 might protect cells from lipotoxicity

• Structural biology of FABP3 complexes:

  • Apply cryo-EM or X-ray crystallography to resolve the structure of FABP3 in complex with its binding partners

  • Investigate the structural basis for FABP3's ability to form higher-order assemblies with inflammatory enzymes

  • Develop structure-based approaches to modulate FABP3 function

• FABP3 in non-canonical tissues:

  • Explore FABP3 functions in tissues where it is expressed at lower levels

  • Investigate tissue-specific binding partners and signaling pathways

  • Study potential redundancy and compensation between FABP family members

These research directions could significantly advance our understanding of FABP3 biology and potentially reveal new therapeutic approaches for inflammatory and metabolic disorders.

How can advances in antibody engineering improve FABP3 detection specificity and sensitivity?

Emerging antibody engineering technologies offer promising approaches to enhance FABP3 detection across research and clinical applications:

• Single-domain antibodies (nanobodies):

  • Advantages for FABP3 detection:

    • Smaller size allows access to epitopes that may be sterically hindered for conventional antibodies

    • Improved tissue penetration for in vivo imaging

    • Greater stability under various assay conditions

  • Application potential: Development of nanobody-based immunoassays with improved sensitivity for low FABP3 concentrations

• Bispecific antibody formats:

  • Design of bispecific antibodies targeting:

    • Two different epitopes on FABP3 for increased specificity

    • FABP3 plus another cardiac biomarker (e.g., troponin) for multiplex detection

  • Benefits: Single-molecule detection of multiple analytes with reduced assay complexity

• Affinity maturation techniques:

  • Application of directed evolution approaches:

    • Phage display with stringent selection conditions

    • Yeast surface display with fluorescence-activated cell sorting

  • Outcome: Development of ultra-high affinity anti-FABP3 antibodies with sub-picomolar binding constants

• Recombinant antibody engineering:

  • Modification of framework regions to enhance:

    • Thermal stability for point-of-care applications

    • Resistance to interfering substances in complex biological samples

    • Extended shelf-life for commercial assays

  • Current examples: Recombinant anti-FABP3 antibodies like EPR22017-202 (capture) and EPR22017-264 (detector) show improved performance characteristics

• Site-specific conjugation strategies:

  • Precise control over conjugation chemistry:

    • Enzymatic approaches (sortase, transglutaminase)

    • Click chemistry with non-canonical amino acids

  • Advantages: Homogeneous antibody-label orientation leading to improved sensitivity and reduced lot-to-lot variability

• Computationally designed antibodies:

  • In silico antibody design targeting FABP3-specific epitopes

  • Structure-based optimization of antibody-antigen interfaces

  • Potential for creating antibodies with predefined properties for specific applications

These advanced antibody engineering approaches hold significant promise for developing next-generation FABP3 detection systems with improved sensitivity, specificity, and versatility across research and clinical applications.