FGF19 Antibody, FITC conjugated

What is the biological function of FGF19 in human physiology?

FGF19 (Fibroblast Growth Factor 19) serves as a physiological regulator of bile acid homeostasis in humans. It functions primarily by suppressing bile acid biosynthesis through down-regulation of CYP7A1 expression, following positive regulation of the JNK and ERK1/2 signaling cascades. Additionally, FGF19 stimulates glucose uptake in adipocytes and has emerged as a potent insulin sensitizer capable of normalizing plasma glucose concentration, improving lipid profiles, and ameliorating fatty liver disease. The activity of FGF19 requires the presence of two co-receptors: KLB (βklotho) and FGFR4 (Fibroblast Growth Factor Receptor 4) .

How can I design experiments to investigate FGF19's dual role in metabolism and cell proliferation?

To investigate FGF19's dual functions in metabolism and cell proliferation, a comprehensive experimental design should include:

• Metabolic assessment:

  • Measure glucose uptake in adipocytes or hepatocytes using glucose uptake assays

  • Analyze bile acid synthesis by measuring expression of CYP7A1 using RT-PCR

  • Perform glucose tolerance tests in animal models

• Cell proliferation analysis:

  • BrdU incorporation assays to measure DNA synthesis rates

  • Cell cycle analysis by flow cytometry

  • Assessment of EGFR and Wnt signaling pathway activation

When using FITC-conjugated FGF19 antibodies, these can be employed to track FGF19 localization during these processes using confocal microscopy. Additionally, comparing wild-type FGF19 with engineered variants that show reduced mitogenic potential (such as FGF19 ΔKLB) can help differentiate between metabolic and proliferative signaling pathways .

What techniques can be combined with FITC-conjugated FGF19 antibody detection for multi-parameter analysis?

For comprehensive multi-parameter analysis using FITC-conjugated FGF19 antibodies, researchers can implement the following combinations:

When designing multi-parameter experiments, ensure spectral compatibility between fluorophores to minimize bleed-through, and include appropriate compensation controls when using flow cytometry .

What is the recommended protocol for using FITC-conjugated FGF19 antibodies in flow cytometry?

For optimal flow cytometry results with FITC-conjugated FGF19 antibodies, follow this protocol:

• Sample preparation:

  • Harvest cells (1-5 × 10^6 cells/sample)

  • Wash twice in cold PBS containing 2% FBS

  • Fix cells with 2% paraformaldehyde for 15 minutes at room temperature (if intracellular staining is required)

  • Permeabilize with 0.1% saponin in PBS for 10 minutes (for intracellular targets only)

• Staining procedure:

  • Block with 2% normal serum from the same species as secondary antibody for 30 minutes

  • Incubate with FITC-conjugated FGF19 antibody at 1-5 μg/mL for 30-45 minutes at 4°C in the dark

  • Wash three times with PBS containing 2% FBS

  • Resuspend in PBS with 2% FBS for immediate analysis

• Controls:

  • Include an isotype control conjugated to FITC

  • Use FGF19-transfected cells as a positive control

  • Use non-transfected cells as a negative control

• Instrument settings:

  • Excitation: 488 nm laser

  • Emission: 530/30 nm bandpass filter

  • Perform compensation if multiple fluorophores are used

How should I optimize antibody concentration for immunofluorescence with FITC-conjugated FGF19 antibodies?

To determine the optimal antibody concentration for immunofluorescence with FITC-conjugated FGF19 antibodies, perform a titration experiment following these guidelines:

• Titration series:

  • Prepare a serial dilution of the antibody (typically 0.1-10 μg/mL)

  • Test each concentration on identical samples with known FGF19 expression

• Evaluation criteria:

  • Signal-to-noise ratio (quantify signal intensity vs. background)

  • Specificity (confirm pattern matches expected subcellular localization)

  • Reproducibility across replicate samples

• Optimization table:

• Validation:

  • Confirm specificity using competing peptide or FGF19 knockout/knockdown samples

  • Verify results across different cell types that express varying levels of FGF19

What are the recommended storage conditions to maintain the activity of FITC-conjugated antibodies?

To preserve the fluorescence activity and binding capacity of FITC-conjugated FGF19 antibodies, adhere to these storage guidelines:

• Short-term storage (up to 1 month):

  • Store at 4°C in the dark

  • Add sodium azide (0.02-0.05%) as a preservative

  • Protect from light using amber vials or by wrapping in aluminum foil

• Long-term storage:

  • Store at -20°C in small aliquots to avoid repeated freeze-thaw cycles

  • Add a cryoprotectant such as glycerol (final concentration 30-50%)

  • Include a protein stabilizer such as BSA (0.1-1%)

• Stability considerations:

  • FITC is pH-sensitive; maintain storage buffer at pH 7.2-7.4

  • FITC conjugates are photosensitive; minimize exposure to light

  • Avoid repeated freeze-thaw cycles (limit to <5) which can cause protein aggregation and fluorophore degradation

• Quality control:

  • Periodically verify activity using flow cytometry or microscopy

  • Discard antibody if significant loss of fluorescence intensity is observed

How can I differentiate between specific binding and autofluorescence when using FITC-conjugated FGF19 antibodies?

Distinguishing specific FITC-conjugated FGF19 antibody binding from autofluorescence requires systematic controls and analytical approaches:

• Essential controls:

  • FITC-conjugated isotype control antibody (same species, isotype, and fluorophore:protein ratio)

  • Unstained samples to establish baseline autofluorescence

  • FGF19-negative samples (knockout or knockdown) as negative controls

  • Pre-absorption with recombinant FGF19 protein to confirm specificity

• Analytical approaches:

  • Spectral unmixing to separate FITC signal from autofluorescence

  • Multi-parameter analysis using antibodies against known FGF19 interacting partners (FGFR4, KLB)

  • Comparison of fluorescence patterns between fixed and unfixed samples

• Technical considerations:

  • Autofluorescence typically has broader emission spectra than FITC

  • Cellular autofluorescence often correlates with cell size/granularity

  • Tissue autofluorescence is often associated with specific structures (lipofuscin, elastin)

• Advanced solutions:

  • Use time-gated detection (FITC has longer fluorescence lifetime than autofluorescence)

  • Consider alternative fluorophores with emission in far-red spectrum if autofluorescence is problematic

What approaches should I take when Western blot results with FGF19 antibodies show unexpected bands?

When encountering unexpected bands in Western blots using FGF19 antibodies, implement this systematic troubleshooting approach:

• Potential causes and solutions:

• Validation strategies:

  • Compare results from multiple anti-FGF19 antibodies targeting different epitopes

  • Test antibody against recombinant FGF19 protein as positive control

  • Include FGF19-transfected cell lysate as reference (expected 24 kDa band)

  • Compare with known FGF19-negative samples

• Technical optimization:

  • Adjust antibody concentration (typically 1 μg/mL for Western blotting)

  • Optimize blocking reagents (5% non-fat milk or 3-5% BSA)

  • Test different membrane types (PVDF vs. nitrocellulose)

  • Consider gradient gels for better separation

How do I analyze and interpret FGF19 signaling pathway activation in experimental data?

When analyzing FGF19 signaling pathway activation from experimental data, follow these systematic approaches:

• Key signaling nodes to assess:

  • FGFR4 and KLB receptor complex formation

  • JNK and ERK1/2 phosphorylation status (early signaling events)

  • CYP7A1 expression levels (metabolic endpoint)

  • EGFR and Wnt/β-catenin pathway activation (proliferative endpoints)

• Integrated data analysis framework:

• Differential activation thresholds:

  • Metabolic effects require weaker FGFR dimerization and transient signaling

  • Proliferative effects require stronger FGFR dimerization and sustained signaling

  • Gene expression changes in BA biosynthesis genes vs. cancer-related genes can distinguish pathway bias

• Comparison with engineered FGF19 variants:

  • Wild-type FGF19 activates both metabolic and proliferative pathways

  • FGF19 ΔFGFR shows reduced proliferative but maintained metabolic signaling

  • FGF19 ΔHBS shows further reduced proliferative with maintained metabolic signaling

  • FGF19 ΔKLB shows minimal proliferative with fully preserved metabolic signaling

How can FITC-conjugated FGF19 antibodies be used to study FGF19-FGFR-KLB-HS quaternary complex formation?

FITC-conjugated FGF19 antibodies provide powerful tools for investigating the complex formation between FGF19, FGFR, KLB, and heparan sulfate (HS) through several advanced methodological approaches:

• Live-cell imaging techniques:

  • Use FITC-conjugated FGF19 antibodies to track FGF19 localization in real-time

  • Combine with differently labeled antibodies against FGFR4 and KLB for co-localization studies

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to assess complex dynamics and stability

• Super-resolution microscopy approaches:

  • STORM or PALM imaging using photoconvertible fluorophores for nanoscale resolution

  • Structured illumination microscopy (SIM) for enhanced spatial resolution of complex components

  • Combination with proximity ligation assays for validation of protein-protein interactions

• Quantitative complex analysis:

  • Implement automated image analysis algorithms to quantify co-localization coefficients

  • Utilize FLIM-FRET (Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer) to measure molecular distances between complex components

  • Correlate with functional readouts such as ERK1/2 phosphorylation using multi-parameter imaging

• Methodological considerations:

  • Optimize antibody concentration to achieve sufficient signal without disrupting native complexes

  • Consider the potential impact of antibody binding on complex formation and stability

  • Validate findings using complementary techniques such as co-immunoprecipitation

What are the current experimental approaches to developing non-mitogenic FGF19 variants for therapeutic applications?

Developing non-mitogenic FGF19 variants while preserving beneficial metabolic effects involves several sophisticated experimental approaches:

• Structure-guided engineering strategies:

  • Targeted mutagenesis of residues involved in FGFR, HS, or KLB binding

  • Examples include FGF19 ΔFGFR (Y115A mutation), FGF19 ΔHBS (K149A mutation), and FGF19 ΔKLB (D198A mutation)

  • Engineering chimeric proteins combining regions from FGF19 and FGF21

• Functional validation pipeline:

• Threshold-based design principles:

  • Exploit differential signaling thresholds for metabolic vs. proliferative pathways

  • Target receptor dimerization strength to maintain weak (metabolic) signaling while eliminating strong (proliferative) signaling

  • Focus on modifications that affect the stability and duration of receptor complexes

• Advanced animal model testing:

  • Chronic administration studies in db/db mice and normal C57BL/6J mice

  • Assessment of both glycemic control and hepatocellular proliferation

  • Monitoring of EGFR/TGFα and Wnt/β-catenin signaling pathways

How can FITC-conjugated FGF19 antibodies be used to study the differential effects of FGF19 variants on metabolic versus proliferative signaling?

FITC-conjugated FGF19 antibodies provide valuable tools for dissecting the distinct signaling pathways activated by different FGF19 variants:

• Cellular localization and trafficking studies:

  • Track the subcellular localization of FGF19 variants in real-time

  • Compare receptor complex formation patterns between variants using multi-color imaging

  • Assess endocytosis and trafficking rates of different FGF19-receptor complexes

• Signaling dynamics assessment:

  • Correlate FGF19 binding with downstream signaling pathway activation

  • Measure the duration and intensity of ERK1/2 phosphorylation following stimulation

  • Implement live-cell reporters for real-time monitoring of signaling pathways

• Multi-parameter approach for pathway discrimination:

• Advanced methodological considerations:

  • Use dual-labeled FGF19 antibodies to track both location and conformation changes

  • Implement correlation spectroscopy techniques to measure diffusion rates of different complexes

  • Combine with CRISPR-based genetic screens to identify differential pathway components

What controls are essential when validating the specificity of FITC-conjugated FGF19 antibodies?

Rigorous validation of FITC-conjugated FGF19 antibodies requires a comprehensive set of controls:

• Positive controls:

  • FGF19-transfected cell lysates (showing expected 24 kDa band in Western blot)

  • Recombinant human FGF19 protein

  • Tissues/cells known to express FGF19 (e.g., ileum, liver)

• Negative controls:

  • Non-transfected cell lysates

  • FGF19 knockout or knockdown samples

  • Tissues/cells known not to express FGF19

• Specificity controls:

  • Pre-absorption with recombinant FGF19 protein

  • Competing peptide controls

  • Cross-reactivity testing with related FGF family members (FGF21, FGF23)

• Technical controls:

  • FITC-conjugated isotype control antibody

  • Secondary antibody-only controls (for indirect methods)

  • Autofluorescence controls (untreated samples)

• Validation matrix:

These controls collectively ensure that the observed signals represent genuine FGF19 detection rather than technical artifacts or cross-reactivity .

How do different fixation and permeabilization methods affect epitope recognition by FGF19 antibodies?

The choice of fixation and permeabilization methods significantly impacts epitope preservation and accessibility for FGF19 antibody detection:

• Fixation methods comparison:

• Permeabilization method effects:

• Optimization strategies:

  • Test multiple fixation/permeabilization combinations for each application

  • Consider epitope location (N-terminal, C-terminal, internal)

  • Adjust incubation times to balance penetration vs. epitope preservation

  • Implement antigen retrieval methods if needed (heat-induced or enzymatic)

What quantitative methods can be used to measure FGF19 levels in biological samples using FITC-conjugated antibodies?

Several quantitative approaches can be employed with FITC-conjugated FGF19 antibodies for accurate measurement in biological samples:

• Flow cytometry-based quantification:

  • Quantitative flow cytometry using calibration beads

  • Measurement of molecules of equivalent soluble fluorochrome (MESF)

  • Single-cell analysis of FGF19 expression levels across populations

• Microscopy-based quantification:

  • Integrated fluorescence intensity measurement

  • Automated high-content imaging with quantitative analysis

  • Ratio imaging using internal reference standards

• Calibration and standardization approaches:

• Data analysis considerations:

  • Background subtraction using appropriate negative controls

  • Correction for autofluorescence contribution

  • Standardization across different instruments and experiments

  • Statistical validation using technical and biological replicates

• Assay validation parameters:

  • Analytical sensitivity: lowest detectable concentration

  • Analytical specificity: lack of interference from related proteins

  • Precision: intra- and inter-assay coefficients of variation

  • Linearity: proportional response across concentration range

  • Recovery: accurate measurement in complex biological matrices

How are FGF19 antibodies being used to study the therapeutic potential of FGF19 in metabolic diseases?

FGF19 antibodies are integral to advancing research on FGF19's therapeutic potential for metabolic diseases through several cutting-edge approaches:

• Therapeutic target validation:

  • Tracking FGF19 distribution and receptor binding in metabolic tissues

  • Correlating FGF19 levels with disease severity in patient samples

  • Monitoring changes in FGF19 signaling during disease progression

• Development of FGF19-based therapeutics:

  • Screening engineered FGF19 variants for optimal metabolic/minimal proliferative activities

  • Comparing wild-type FGF19 with variants like FGF19 ΔKLB that maintain beneficial metabolic effects

  • Validating efficacy of FGF19 analogs in normalizing blood glucose and regulating bile acid synthesis

• Mechanism elucidation:

  • Dissecting the differential signaling thresholds between metabolic and proliferative pathways

  • Identifying tissue-specific responses to FGF19 signaling

  • Characterizing the interplay between FGF19 and other metabolic regulators

• Clinical translation research:

  • Developing companion diagnostics to identify optimal responders to FGF19-based therapies

  • Monitoring on-target and off-target effects during clinical trials

  • Assessing long-term safety through detection of proliferative marker changes

• Potential therapeutic applications:

Research with these antibodies is revealing that fine-tuning of receptor dimerization and downstream signaling thresholds provides a practical approach for engineering safer FGF19 agonists for treating metabolic diseases .

What recent advances have been made in understanding the structural basis of FGF19's dual metabolic and proliferative functions?

Recent structural biology advances have significantly enhanced our understanding of FGF19's bifunctional nature:

• Quaternary complex structure insights:

  • Detailed 2:2:2:2 FGF19-FGFR1c-KLB-HS complex model reveals coordination between multiple binding partners

  • Critical residues that mediate FGF19-FGFR interactions identified (e.g., Tyr-115)

  • Structure of the atypical heparan sulfate (HS) binding site elucidated (involving Lys-149)

  • C-terminal tail interactions with KLB mapped (with Asp-198 playing a key role)

• Structure-function correlation:

  • Residues critical for FGFR dimerization identified through mutagenesis and functional studies

  • Differential binding interfaces associated with metabolic vs. proliferative outcomes characterized

  • Conformational changes induced by receptor binding correlated with downstream signaling bias

• Advanced structural techniques employed:

• Translational outcomes:

  • Rational design of FGF19 variants with selective pathway activation

  • Development of the threshold model for FGF signaling specificity

  • Creation of variants with progressively reduced dimerization capacity (FGF19 ΔFGFR, FGF19 ΔHBS, FGF19 ΔKLB)

  • Demonstration that metabolic signaling requires weaker receptor dimerization than proliferative signaling

How might single-cell analysis technologies using FITC-conjugated FGF19 antibodies advance our understanding of heterogeneous responses to FGF19?

Emerging single-cell technologies combined with FITC-conjugated FGF19 antibodies offer unprecedented insights into cellular heterogeneity in FGF19 responses:

• Advanced single-cell methodologies:

  • Single-cell RNA-seq combined with protein detection (CITE-seq) to correlate FGF19 binding with transcriptional responses

  • Mass cytometry (CyTOF) with metal-labeled antibodies for high-parameter analysis of FGF19 signaling

  • Single-cell Western blotting to detect FGF19-induced signaling in individual cells

  • Spatial transcriptomics to map FGF19 responses within tissue architecture

• Key biological questions addressable:

  • Cell-to-cell variability in FGF19 receptor expression and signaling responses

  • Identification of distinct cellular subpopulations with differential sensitivity to FGF19

  • Transition dynamics between metabolic and proliferative states

  • Spatial organization of FGF19 responsive cells within complex tissues

• Technological integration approaches:

• Translational implications:

  • Identification of cellular biomarkers predictive of FGF19 therapeutic response

  • Understanding mechanisms of resistance to FGF19-based therapies

  • Development of targeted combination approaches based on cell-specific response patterns

  • Precision medicine strategies for metabolic disease treatment

What are the best practices for selecting and validating FGF19 antibodies for specific research applications?

When selecting and validating FGF19 antibodies, researchers should implement these comprehensive best practices:

• Application-specific selection criteria:

  • Western blot: Select antibodies validated for denatured epitopes

  • Immunofluorescence: Choose antibodies validated for preserved cellular morphology

  • Flow cytometry: Prefer directly conjugated antibodies for single-step detection

  • Functional studies: Select non-neutralizing antibodies that don't interfere with FGF19 activity

• Validation framework:

  • Multi-technique validation using orthogonal methods

  • Testing across multiple biological contexts (different cell types/tissues)

  • Inclusion of appropriate positive and negative controls

  • Comparison with alternative antibodies targeting different epitopes

• Documentation and reporting standards:

  • Record complete antibody information (catalog number, lot, concentration used)

  • Document validation experiments in detail

  • Include all controls in published research

  • Share validation data with research community

• Decision-making matrix for antibody selection:

Following these best practices ensures reliable, reproducible research outcomes and facilitates comparison across different studies investigating FGF19 biology and therapeutic applications .

What are the key considerations for researchers designing experiments to study FGF19 signaling specificity?

Researchers investigating FGF19 signaling specificity should incorporate these critical experimental design elements:

• Receptor complex considerations:

  • Account for the quaternary complex (FGF19-FGFR-KLB-HS) in experimental design

  • Verify expression levels of all components in model systems

  • Consider tissue-specific variations in receptor/co-receptor distribution

  • Address the impact of receptor dimerization strength on signaling outcomes

• Signaling dynamics approach:

  • Implement time-course experiments to distinguish transient from sustained signaling

  • Measure both early (minutes to hours) and late (hours to days) signaling events

  • Compare dose-response relationships across different pathways

  • Correlate receptor dimerization strength with pathway activation thresholds

• Pathway discrimination strategy:

• Comparative approach using FGF19 variants:

  • Include wild-type FGF19 as reference standard

  • Test engineered variants with defined receptor binding characteristics

  • Compare variants with selective metabolic vs. proliferative activities

  • Implement parallel in vitro and in vivo experimental systems

• Technical and biological controls:

  • Include receptor/co-receptor knockdown controls

  • Implement pathway-specific inhibitors as reference points

  • Use FGF19 ΔKLB as a metabolic-only positive control

  • Include FGF19 ΔC-tail as negative control unable to bind KLB