Fgf2 Antibody， Biotin conjugated

What is FGF2 and what are its primary biological functions?

FGF2 (Fibroblast Growth Factor 2), also known as basic fibroblast growth factor (bFGF) or heparin-binding growth factor 2 (HBGF-2), is a multifunctional growth factor with diverse biological activities. It acts as a ligand for multiple fibroblast growth factor receptors including FGFR1, FGFR2, FGFR3, and FGFR4. Beyond receptor binding, FGF2 functions as an integrin ligand, specifically binding to integrin ITGAV:ITGB3, which is required for FGF2 signaling. FGF2 plays crucial roles in regulating cell survival, cell division, cell differentiation, and cell migration. In vitro studies have demonstrated that it functions as a potent mitogen. Additionally, FGF2 can induce angiogenesis and mediates phosphorylation of ERK1/2, which promotes retinal lens fiber differentiation .

What are the key characteristics of biotin-conjugated FGF2 antibodies?

Biotin-conjugated FGF2 antibodies, such as Rabbit Polyclonal FGF2 antibody-biotin (ab84027), are immunological tools designed for specific detection of FGF2 in various research applications. These antibodies are typically generated against recombinant full-length human FGF2 protein and validated for applications including Western Blotting (WB) and sandwich ELISA (sELISA). The biotinylation process enables enhanced detection sensitivity through streptavidin-based detection systems, leveraging the high-affinity biotin-streptavidin interaction. This characteristic makes these antibodies particularly valuable for detecting even low levels of FGF2 expression in experimental samples .

What experimental applications are biotin-conjugated FGF2 antibodies validated for?

Based on the available information, biotin-conjugated FGF2 antibodies have been validated for Western Blotting (WB) and sandwich ELISA (sELISA) applications specifically with human samples . The biotin conjugation provides significant advantages in these applications by enabling signal amplification through streptavidin-based detection systems. For Western blotting, these antibodies can detect FGF2 protein in complex biological samples after separation by gel electrophoresis. In sandwich ELISA, the biotin conjugation facilitates sensitive quantitative detection of FGF2 in solution through high-affinity interactions with streptavidin-conjugated reporter molecules. These applications are particularly valuable for studying FGF2 expression and quantification in research contexts.

How can researchers assess FGF2-receptor interactions using biotin-conjugated systems?

To evaluate FGF2-receptor interactions using biotin-conjugated systems, researchers can employ several methodological approaches. Biolayer interferometry (BLI) represents an effective technique as demonstrated in published research. For this approach, biotinylated extracellular domains of FGFR1c fused to Fc fragments are immobilized on streptavidin biosensors, followed by exposure to wild-type FGF2 or FGF2 conjugates . This method allows real-time monitoring of binding kinetics and determination of dissociation constants (Kd).

Another approach involves phosphorylation assays to assess downstream signaling activation. Researchers can treat cells expressing FGFRs (e.g., NIH 3T3 cells) with FGF2 for 15 minutes and then analyze activation of signaling pathways through Western blotting with anti-phospho-ERK 1/2 antibodies and anti-phospho-FGFR1 antibodies . This approach verifies whether FGF2 variants maintain their ability to stimulate downstream signaling at levels comparable to wild-type FGF2.

For cell-based receptor binding studies, flow cytometry can be employed using biotin-conjugated FGF2, followed by streptavidin-fluorophore detection. This enables quantification of FGF2 binding to cell surface receptors and comparison across different cell lines with varying receptor expression levels.

What protocols can be optimized for Western blotting using biotin-conjugated FGF2 antibodies?

When optimizing Western blotting protocols with biotin-conjugated FGF2 antibodies, researchers should consider several critical parameters for optimal results:

• Sample preparation: Use complete protease inhibitor cocktails during protein extraction to preserve FGF2 integrity, as it can be sensitive to proteolytic degradation.

• Gel electrophoresis: 10-12% SDS-PAGE gels are typically suitable for resolving FGF2 (~18-24 kDa depending on isoform).

• Membrane selection: PVDF membranes may provide better protein retention than nitrocellulose for FGF2 detection.

• Blocking strategy: Use BSA-based blockers (3-5%) rather than milk-based blockers, as milk contains endogenous biotin that might interfere with detection.

• Antibody incubation: Optimize both concentration (typically starting at 1:1000 dilution) and incubation time (4°C overnight often yields best results).

• Detection system: Employ streptavidin conjugated to HRP, fluorophores, or other detection molecules. For enhanced sensitivity, consider amplification systems like avidin-biotin complex (ABC) method.

• Controls: Include appropriate positive (recombinant FGF2) and negative controls to validate detection specificity.

What methodological considerations are important for sandwich ELISA using biotin-conjugated FGF2 antibodies?

When implementing sandwich ELISA with biotin-conjugated FGF2 antibodies, researchers should optimize several key aspects of the protocol:

• Antibody selection: Choose a capture antibody that recognizes a different epitope than the biotin-conjugated detection antibody to prevent competitive binding.

• Plate coating: Coat high-binding 96-well plates with capture antibody (typically 1-5 μg/mL) in carbonate/bicarbonate buffer (pH 9.6) overnight at 4°C.

• Blocking strategy: Use protein-free blocking buffer to minimize background while avoiding biotin-containing blocking agents like milk.

• Sample preparation: Employ sample diluents that minimize matrix effects while preserving FGF2 immunoreactivity.

• Standard curve: Prepare a dilution series using recombinant FGF2 (typically 0-1000 pg/mL) to establish assay linearity and sensitivity.

• Detection system: Apply the biotin-conjugated FGF2 antibody at optimized concentration followed by streptavidin-HRP conjugate. Develop with appropriate substrate and measure spectrophotometrically.

• Validation: Include controls for non-specific binding and confirm antibody specificity through spike-and-recovery and parallelism assessments.

How can FGF2-based systems be utilized in developing targeted cancer therapeutics?

The published research reveals sophisticated applications of FGF2 in developing targeted cancer therapeutics, particularly through the creation of FGF2-drug conjugates. Researchers have developed novel protein-drug conjugates using FGF2 as a targeting molecule for cancers overexpressing FGFR1. One advanced approach involves conjugating the cytotoxic agent monomethyl auristatin E (MMAE) to FGF2, creating conjugates that specifically target and kill FGFR1-positive cancer cells .

These FGF2-MMAE conjugates effectively target FGFR1-expressing cells, undergo FGFR1-mediated endocytosis, and exhibit cytotoxicity that correlates with FGFR1 expression levels . Studies have demonstrated that increasing the number of MMAE molecules attached to FGF2 (from 1 to 3) enhanced cytotoxic potency, highlighting the importance of optimizing drug loading.

For improved pharmacokinetic properties, advanced conjugates incorporating hydrophilic PEG4 or PEG27 spacers between FGF2 and MMAE have been developed. The conjugate with the highest hydrodynamic size (42 kDa) demonstrated superior cytotoxicity and effectively inhibited tumor growth in a mouse model of human breast cancer . This approach represents a promising alternative to antibody-drug conjugates for targeting FGFR1-overexpressing tumors.

What methodologies exist for site-specific conjugation to FGF2?

Site-specific conjugation to FGF2 can be achieved through several advanced methodological approaches:

• Unnatural amino acid incorporation: Researchers have successfully incorporated propargyllysine (PrK) into FGF2 using amber codon suppression technology. The alkyne moiety in PrK enables highly specific conjugation through Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC) with azide-functionalized molecules .

• Cysteine-directed conjugation: FGF2 contains four cysteines, two of which are highly exposed and amenable to conjugation. Researchers have created FGF2 variants with controlled reactivity by selectively mutating specific cysteines (C78S/C96S mutations) while preserving others for targeted modification .

• Terminal extensions: N- and C-terminal extensions containing cysteine residues (such as KCK tags) have been engineered into FGF2 to provide additional conjugation sites without disrupting the protein's core structure and biological activity .

• Reaction optimization: For challenging conjugations, particularly with hydrophobic molecules, detergent-based approaches have demonstrated improved efficiency, achieving nearly 100% yield in difficult cases .

These methodological advances enable the creation of defined, homogeneous FGF2 conjugates with preserved biological activity for various research and therapeutic applications.

How does FGF2 maintain biological activity after conjugation?

The impact of conjugation on FGF2's biological activity depends on the conjugation strategy and modification sites. According to published studies, carefully designed FGF2 conjugates maintain their ability to bind receptors and activate downstream signaling. Research has demonstrated that FGF2 variants with cysteine-to-serine substitutions and N/C-terminal extensions, as well as their conjugates with vcMMAE, stimulated downstream signaling at levels comparable to wild-type FGF2, as evidenced by phosphorylation of ERK 1/2 and FGFR1 .

Biolayer interferometry (BLI) analysis has confirmed that FGF2 conjugates retained binding to the recombinant extracellular domain of FGFR1c, demonstrating that the introduced modifications did not impair receptor recognition . Additionally, fluorescently labeled FGF2 conjugates demonstrated preserved cellular internalization via FGFR1-mediated endocytosis, indicating functional retention of this critical biological property.

The data suggests that FGF2 is more robust than some other growth factors (like FGF1) in maintaining stability after modification. FGF2's higher resistance to thermal unfolding, aggregation, and proteolysis allows it to better tolerate conjugation while preserving functionality . This intrinsic stability makes FGF2 particularly valuable as a targeting molecule for bioconjugation strategies in research and therapeutic applications.

What factors affect the cytotoxicity of FGF2-cytotoxic drug conjugates in FGFR1-expressing cells?

Several critical factors influence the cytotoxicity of FGF2-cytotoxic drug conjugates in FGFR1-expressing cells:

• Drug-to-protein ratio (DPR): Research demonstrates that conjugates bearing three monomethyl auristatin E (MMAE) molecules exhibited higher cytotoxicity than those with fewer attached drug molecules, confirming a direct relationship between drug loading and in vitro cytotoxicity .

• FGFR1 expression level: Cytotoxicity studies across multiple cell lines revealed a clear correlation between FGFR1 expression and sensitivity to FGF2-MMAE conjugates. Cells with high FGFR1 expression (U2OS-R1) showed significantly greater sensitivity than those with low or undetectable FGFR1 levels (U2OS), demonstrating the targeting specificity of these conjugates .

• Internalization efficiency: Confocal microscopy studies confirmed that FGF2 conjugates were effectively internalized through FGFR1-mediated endocytosis. This internalization is essential for the release of the active cytotoxic agent within target cells .

• Linker chemistry: The valine-citrulline (vc) linker used in FGF2-vcMMAE conjugates enables specific cleavage by intracellular cathepsins following endocytosis, releasing active MMAE inside the cell. Linker design must balance stability in circulation with efficient drug release in target cells .

• Hydrodynamic size: Among conjugates studied, those with higher hydrodynamic size (42 kDa) exhibited optimal in vivo efficacy in a mouse model of human breast cancer, suggesting that size optimization can enhance therapeutic outcomes .

These factors provide important considerations for researchers designing and optimizing FGF2-based targeted therapeutics.

How can researchers optimize conjugation reactions for creating FGF2 conjugates?

Optimizing conjugation reactions for FGF2 conjugates requires systematic evaluation of multiple parameters:

• Site-specific strategy selection:

  • For cysteine-directed conjugation using maleimide chemistry, buffer should be PBS pH 7.2-7.4 with 1-5 mM EDTA to prevent disulfide formation

  • For click chemistry (CuAAC) with unnatural amino acids like propargyllysine, consider Cu(I) catalyst concentration, Cu(I)-stabilizing ligands, and mild detergents

• Reaction conditions optimization:

  • Molar ratio of conjugation reagent to protein (typically 5-20 fold molar excess)

  • Reaction temperature and time (often 4°C overnight balances efficiency with protein stability)

  • For hydrophobic molecules, addition of mild detergents (0.05-0.1% Tween-20 or Triton X-100) improves solubility and reaction efficiency

• Purification strategy:

  • Size exclusion chromatography to remove unreacted small molecules

  • Additional chromatographic steps for higher purity if needed

• Quantification methods:

  • Degree of conjugation should be quantified using methods such as mass spectrometry or spectrophotometric assays

  • Functional activity assays to ensure biological function is preserved

Published research demonstrates that optimizing these parameters can achieve nearly 100% conjugation efficiency even for challenging reactions with hydrophobic compounds like MMAE .

What analytical methods confirm successful conjugation to FGF2?

Researchers can employ several analytical techniques to verify successful conjugation to FGF2:

• Mass spectrometry: MALDI-TOF or ESI-MS analysis reveals mass shifts corresponding to the addition of conjugated molecules. LC-MS/MS following proteolytic digestion can identify specific modified residues with high precision.

• SDS-PAGE with appropriate detection: For biotin conjugates, samples can be analyzed by gel electrophoresis followed by detection with streptavidin-HRP or streptavidin-fluorophore conjugates. Band shifts may be observed with multiple additions.

• Functional binding assays: Biolayer interferometry (BLI) studies have been successfully employed to confirm that FGF2 conjugates maintain binding to recombinant FGFR1c extracellular domains, verifying that the conjugation did not disrupt receptor recognition .

• Cell-based assays: Confocal microscopy using fluorescently labeled FGF2 conjugates has demonstrated preserved cellular internalization via FGFR1-mediated endocytosis, confirming functional integrity .

• Signaling pathway activation: Western blotting with anti-phospho-ERK 1/2 antibodies and anti-phospho-FGFR1 has been used to verify that FGF2 conjugates maintain the ability to stimulate downstream signaling pathways, confirming biological activity post-conjugation .

These complementary approaches provide comprehensive verification of both the chemical success of conjugation and the preservation of biological function.

How do FGF2-based therapeutic approaches compare to other targeted strategies?

FGF2-based therapeutic approaches offer several distinctive advantages and limitations compared to other targeted strategies:

Research has demonstrated that FGF2-MMAE conjugates effectively target FGFR1-expressing cells and exhibit cytotoxicity that correlates with FGFR1 expression levels . The incorporation of hydrophilic PEG spacers has further improved pharmacokinetic properties, with conjugates of optimal hydrodynamic size (42 kDa) showing effective inhibition of tumor growth in mouse models of human breast cancer .

These findings position FGF2-based approaches as promising alternatives to antibody-drug conjugates, particularly for targeting cancers with FGFR1 overexpression, where they may offer advantages in manufacturing simplicity, cost, and tumor penetration.

What methodological considerations exist for evaluating FGF2 conjugate internalization?

Evaluating FGF2 conjugate internalization requires careful methodological design:

• Fluorescent labeling strategies:

  • Published research has successfully employed DyLight 550-labeled FGF2 conjugates to track cellular uptake via confocal microscopy

  • Dual labeling approaches can simultaneously track both the FGF2 carrier and its conjugated cargo

• Cell model selection:

  • Comparative analysis using cell lines with varying FGFR1 expression levels provides critical controls

  • Co-culture experiments with prestained cell populations (e.g., CellTrace Violet) allow direct comparison on the same experimental platform

• Time-course analysis:

  • Tracking internalization kinetics at multiple timepoints provides information on uptake rates

  • Pulse-chase experimental designs can distinguish surface-bound from internalized conjugates

• Colocalization studies:

  • Simultaneous visualization of FGF2 conjugates with endosomal/lysosomal markers confirms trafficking through expected compartments

  • Quantitative colocalization analysis provides objective measurement of internalization efficiency

• Biochemical fractionation:

  • Cell surface biotinylation followed by streptavidin pull-down can biochemically separate surface-bound from internalized conjugates

  • Subcellular fractionation allows quantitative distribution analysis across cellular compartments

These methodological approaches collectively provide comprehensive assessment of FGF2 conjugate internalization, both qualitatively and quantitatively, enabling researchers to optimize conjugate design for efficient cellular delivery.

How should researchers interpret results from different FGF2-receptor interaction assays?

When researchers encounter results from different FGF2-receptor interaction assays, systematic interpretation should consider several key factors:

• Methodological differences:

  • Cell-free binding assays (BLI, SPR) measure direct physical interactions under controlled conditions

  • Cell-based assays incorporate additional complexities including co-receptors, extracellular matrix, and cellular trafficking

  • Phosphorylation assays measure downstream signaling rather than direct binding

• The critical role of co-factors:

  • Heparan sulfate proteoglycans (HSPGs) function as essential co-receptors stabilizing FGF2-FGFR binding

  • Presence or absence of these co-factors between assay systems may cause apparent discrepancies in affinity measurements

• Experimental conditions:

  • Buffer composition, pH, temperature, and ion concentrations significantly impact FGF2-receptor interactions

  • Physiological calcium concentrations affect FGFR dimerization and subsequent signaling

• Receptor isoform specificity:

  • FGF2 interacts differentially with FGFR1c, FGFR3c, and FGFR4

  • Splice variants within these receptor families show distinct binding characteristics

The most robust interpretation comes from integrating multiple complementary approaches. For example, published research has employed both direct binding studies (BLI) and functional signaling assays (phosphorylation of ERK1/2 and FGFR1) to comprehensively characterize FGF2 conjugates . This multi-method approach provides more complete understanding of both physical binding properties and functional consequences of FGF2-receptor interactions.