FGF4 Antibody

What is FGF4 and what are its biological functions?

FGF4 (Fibroblast Growth Factor 4) is a crucial protein that plays significant roles in embryonic development, cell proliferation, differentiation, and survival. It is required for normal limb and cardiac valve development during embryogenesis. FGF4 may also play a role in embryonic molar tooth bud development by inducing the expression of MSX1, MSX2, and MSX1-mediated expression of SDC1 in dental mesenchyme cells . The protein is involved in regulating neural progenitor cell proliferation and neuronal differentiation, and has been identified by its strong oncogenic transforming activity. It functions as a potent angiogenic factor and is expressed in several highly vascularized tumors as well as in adult mouse testis, intestine, and brain .

What types of FGF4 antibodies are available for research applications?

Several types of FGF4 antibodies are available for research, including:

• Polyclonal antibodies: Most commonly raised in rabbits and recognizing multiple epitopes on the FGF4 protein

• Monoclonal antibodies: Such as mouse monoclonal antibody 2D7D5, which targets specific epitopes with high specificity

• Region-specific antibodies: Targeting different regions of FGF4 including:

  • Middle region (AA 84-97) antibodies

  • C-terminal region antibodies

  • Internal region antibodies

  • N-terminal region antibodies

These antibodies are available in various formats, including unconjugated forms for standard applications or conjugated forms for specialized detection methods .

What are the standard applications for FGF4 antibodies?

FGF4 antibodies can be used for multiple research applications depending on their specific design and validation:

• Western Blotting (WB): For detecting FGF4 protein in cell or tissue lysates with predicted band size of approximately 22 kDa

• Immunohistochemistry (IHC): For detecting FGF4 in tissue sections including paraffin-embedded samples (IHC-P)

• Immunocytochemistry (ICC)/Immunofluorescence (IF): For cellular localization studies

• ELISA: For quantitative measurement of FGF4 in solution

• Neutralization assays: Some antibodies have neutralizing activity against FGF4 biological function

Most antibodies have been tested for specific applications and species reactivity, with human, mouse, and rat being the most common species where cross-reactivity has been confirmed .

How should I select the appropriate FGF4 antibody for my specific experimental application?

The selection of an appropriate FGF4 antibody depends on several factors that should be carefully considered:

• Target species: Confirm the antibody has been validated in your species of interest. For example, antibodies like ABIN3043756 have been tested in human, mouse, and rat samples , while others may have more limited species reactivity.

• Application compatibility: Select antibodies specifically validated for your application:

  • For Western blotting: Antibodies like ab65974 and ab106355 have been validated at concentrations of 0.5-1 μg/mL

  • For IHC: Antibodies such as AF235 have been validated at 15 μg/mL on paraffin-embedded tissue sections

  • For ELISA: Consider antibodies validated in sandwich ELISA formats (0.5-2.0 μg/mL concentration range)

• Epitope location: Different experimental questions may require antibodies targeting specific regions:

  • Middle region antibodies for detecting the core functional domain

  • C-terminal antibodies for studying processing events

  • Full-length antibodies for general detection

• Clonality considerations:

  • Polyclonal antibodies provide higher sensitivity through multiple epitope recognition

  • Monoclonal antibodies offer greater specificity and batch consistency

• Validation evidence: Review immunoblots, IHC images, or neutralization data provided by manufacturers to confirm antibody performance .

What are the recommended protocols for using FGF4 antibodies in Western blotting?

For optimal Western blotting results with FGF4 antibodies, follow these methodological guidelines:

• Sample preparation:

  • Use appropriate cell lines known to express FGF4 (e.g., HeLa cells, NIH 3T3 cells)

  • Prepare complete cell lysates using standard lysis buffers containing protease inhibitors

  • Load approximately 30 μg of total protein per lane

• Antibody concentration:

  • Primary antibody: Use at 0.1-2.0 μg/mL depending on the specific antibody

    • For ab65974: 1 μg/mL

    • For ab106355: 0.5-1 μg/mL

    • For 500-P158: 0.1-0.2 μg/mL

  • Secondary antibody: Use according to manufacturer's recommendations, typically at 1:1000-1:5000 dilution

• Expected results:

  • Predicted band size: 22 kDa for human FGF4

  • Detection limit: Approximately 1.5-3.0 ng/lane of recombinant FGF4 under both reducing and non-reducing conditions

• Controls:

  • Positive control: HeLa or NIH 3T3 cell lysates

  • Negative control: Cell lines with known low FGF4 expression

  • Blocking peptide control: To confirm antibody specificity

• Troubleshooting:

  • If multiple bands appear, optimize blocking conditions or antibody concentration

  • For weak signals, increase antibody concentration or protein loading

  • Consider enhanced chemiluminescence for increased sensitivity

What are the optimal conditions for immunohistochemical detection of FGF4?

For successful immunohistochemical detection of FGF4 in tissue samples:

• Sample preparation:

  • Fix tissues appropriately (typically 10% neutral buffered formalin)

  • For paraffin-embedded sections, perform heat-induced epitope retrieval using antigen retrieval reagent (basic pH buffer is recommended)

  • Section thickness: 4-6 μm is optimal for most applications

• Staining protocol:

  • Blocking: Use serum-free protein block to reduce background

  • Primary antibody:

    • Concentration: 15 μg/mL for AF235 (optimize for other antibodies)

    • Incubation: Overnight at 4°C for optimal results

  • Detection system: HRP-DAB or similar chromogenic detection system

  • Counterstain: Hematoxylin provides good nuclear contrast

• Controls:

  • Positive tissue control: Human breast cancer tissue has been validated

  • Negative control: Omit primary antibody

  • Isotype control: Use non-specific IgG at the same concentration

• Recommended visualization:

  • For bright-field microscopy: HRP-DAB system yields brown staining of target

  • For fluorescence: Use appropriate fluorophore-conjugated secondary antibodies

• Troubleshooting:

  • For high background: Increase blocking time or reduce antibody concentration

  • For weak signal: Optimize antigen retrieval or increase antibody concentration

  • For non-specific binding: Increase washing steps or use more stringent blocking

How can FGF4 antibodies be used to study FGF4 signaling pathways in developmental biology?

FGF4 antibodies are valuable tools for investigating developmental signaling pathways through several advanced approaches:

• Developmental pathway analysis:

  • Immunolocalization studies can track spatial and temporal expression of FGF4 during embryonic development, particularly in limb bud formation and cardiac valve development

  • Co-immunoprecipitation with FGF4 antibodies can identify protein interaction partners in signaling complexes

  • Chromatin immunoprecipitation (ChIP) assays using antibodies against transcription factors regulated by FGF4 can map genomic targets

• Neutralization studies:

  • FGF4 neutralizing antibodies like AF235 can block FGF4 activity (ND₅₀ typically 0.3-0.9 μg/mL)

  • This approach allows for temporal inhibition of FGF4 signaling at specific developmental stages

  • Cell proliferation assays with neutralizing antibodies can quantify the contribution of FGF4 to proliferative responses

• Pathway cross-talk investigation:

  • Combined use of FGF4 antibodies with antibodies against sonic hedgehog (SHH) pathway components can elucidate their interaction in bone morphogenesis and limb development

  • Multiplex immunofluorescence with FGF4 antibodies and markers of MSX1/MSX2 expression can illuminate tooth bud development mechanisms

• Stem cell differentiation studies:

  • Track FGF4 expression during embryonic stem cell differentiation using immunocytochemistry

  • Correlate FGF4 expression with neural progenitor cell proliferation and differentiation markers

  • Study the role of FGF4 in feedback inhibition mechanisms through antibody-based detection of novel FGF4 splice variants

What are the considerations for using FGF4 antibodies in cancer research?

FGF4 antibodies offer valuable tools for cancer research, with several important considerations:

• Tumor expression profiling:

  • FGF4 is expressed in several highly vascularized tumors

  • IHC using antibodies like AF235 at 15 μg/mL has been validated for detection in human breast cancer tissue

  • Western blotting of tumor lysates can quantify expression levels compared to normal tissues

• Angiogenesis studies:

  • As FGF4 is a potent angiogenic factor, antibodies can be used to:

    • Localize FGF4 at sites of tumor vascularization

    • Correlate FGF4 expression with microvessel density

    • Evaluate the effectiveness of anti-angiogenic therapies on FGF4 expression

• Functional studies in cancer models:

  • Neutralizing antibodies (e.g., AF235) can block FGF4-mediated proliferation in cell models

  • Typical neutralization dose (ND₅₀) is 0.3-0.9 μg/mL in the presence of 0.5 ng/mL recombinant human FGF4

  • Combined neutralization of multiple FGF family members may be necessary to fully block pathway activation

• Biomarker development:

  • Sandwich ELISA using FGF4 antibodies can detect as little as 0.2-0.4 ng/well of recombinant human FGF4

  • This sensitivity makes FGF4 antibodies suitable for developing cancer biomarker assays

  • Correlation with clinical outcomes requires carefully validated antibodies with high specificity

• Therapeutic target validation:

  • Antibodies that target specific functional domains of FGF4 can help validate its potential as a therapeutic target

  • Region-specific antibodies (middle region, C-terminal) can help determine which domains are most accessible and functionally important in cancer contexts

How can I optimize FGF4 antibody-based detection in low-expressing samples?

For detecting FGF4 in samples with low expression levels, several optimization strategies can be employed:

• Signal amplification techniques:

  • Tyramide signal amplification (TSA) can increase detection sensitivity up to 100-fold for IHC/ICC applications

  • Polymer-based detection systems provide higher sensitivity than traditional ABC methods

  • Consider using higher antibody concentrations (e.g., 1-2 μg/mL instead of 0.5 μg/mL) for Western blotting

• Sample enrichment approaches:

  • Immunoprecipitation using FGF4 antibodies prior to Western blotting can concentrate the target protein

  • Subcellular fractionation may help concentrate FGF4 in specific cellular compartments

  • Optimized lysis buffers with phosphatase and protease inhibitors can preserve post-translational modifications that might affect antibody recognition

• Enhanced detection methods:

  • For Western blotting, highly sensitive chemiluminescent substrates can detect down to picogram levels

  • For ELISA, the validated sandwich ELISA approach can detect at least 0.2-0.4 ng/well of recombinant human FGF4

  • Digital imaging and analysis software can help quantify weak signals objectively

• Antibody selection considerations:

  • Polyclonal antibodies may provide better sensitivity for low-expressing samples due to recognition of multiple epitopes

  • Middle region antibodies (AA 84-97) targeting conserved functional domains may offer better detection across species

  • Antibodies validated specifically for the application of interest will yield more reliable results

• Technical optimization:

  • Extended primary antibody incubation (overnight at 4°C instead of 1-2 hours at room temperature)

  • Reduced washing stringency to preserve weak signals

  • Optimization of blocking conditions to minimize background while preserving specific signals

How can I validate the specificity of my FGF4 antibody?

Validating antibody specificity is crucial for obtaining reliable research results. For FGF4 antibodies, consider these validation strategies:

• Positive and negative controls:

  • Positive controls: Use cell lines with known FGF4 expression (HeLa, NIH 3T3)

  • Negative controls:

    • Use cell lines with no or low FGF4 expression

    • Perform antibody incubation with the immunizing peptide to block specific binding

    • Include isotype controls at equivalent concentrations

• Molecular weight verification:

  • Confirm detection at the expected molecular weight (22 kDa for human FGF4)

  • Be aware of potential post-translational modifications that might alter apparent molecular weight

  • If multiple bands appear, investigate whether they represent specific isoforms or splice variants

• Genetic validation approaches:

  • CRISPR/Cas9 knockout of FGF4 should eliminate specific antibody signal

  • siRNA knockdown should reduce signal intensity proportional to knockdown efficiency

  • Overexpression systems should show increased signal intensity

• Cross-reactivity assessment:

  • Test for predicted cross-reactivity with related FGF family members

  • Antibodies like ABIN3043756 are reported to have no cross-reactivity with other proteins

  • For species cross-reactivity, test on samples from target species even when predicted to work based on sequence homology

• Application-specific validation:

  • For IHC: Compare staining patterns with published literature and RNA expression data

  • For WB: Molecular weight and band pattern should be consistent with predicted protein

  • For neutralizing antibodies: Confirm functional inhibition in bioassays

What are common issues when using FGF4 antibodies and how can they be resolved?

Researchers may encounter several challenges when working with FGF4 antibodies. Here are common issues and their solutions:

• Non-specific bands in Western blotting:

  • Issue: Detection of multiple unexpected bands

  • Solutions:

    • Optimize antibody concentration (0.1-1 μg/mL is typically recommended)

    • Increase blocking time or change blocking agent

    • Use more stringent washing conditions

    • Consider a different antibody targeting a different epitope

• Weak or no signal:

  • Issue: Inability to detect FGF4 despite expected expression

  • Solutions:

    • Confirm FGF4 expression in your sample by RT-PCR

    • Optimize protein extraction method for your specific sample type

    • Increase antibody concentration or incubation time

    • For Western blotting, load more protein (30 μg or more per lane)

    • For IHC, optimize antigen retrieval methods

• High background in immunostaining:

  • Issue: Non-specific staining obscuring specific signals

  • Solutions:

    • Increase blocking time or concentration

    • Dilute primary antibody appropriately (e.g., 15 μg/mL for IHC with AF235)

    • Extend washing steps

    • Use more specific detection systems

    • Consider using more specific monoclonal antibodies

• Inconsistent results between experiments:

  • Issue: Variable detection between replicates

  • Solutions:

    • Standardize sample preparation and experimental conditions

    • Prepare larger batches of antibody dilutions to reduce variation

    • Include loading controls and quantitative standards

    • Consider antibody lot-to-lot variations and maintain records

• Cross-reactivity issues:

  • Issue: Antibody detects related proteins

  • Solutions:

    • Use antibodies specifically validated for "no cross-reactivity with other proteins"

    • Employ peptide competition assays to confirm specificity

    • Consider using multiple antibodies targeting different epitopes to confirm results

How should FGF4 antibodies be stored and handled to maintain optimal performance?

Proper storage and handling of FGF4 antibodies is essential for maintaining their functionality and extending their usable lifespan:

• Long-term storage recommendations:

  • Temperature: Most antibodies should be stored at -20°C for long-term storage

  • Avoid repeated freeze-thaw cycles by aliquoting antibodies into single-use volumes

  • Protect from light, especially fluorophore-conjugated antibodies

  • Follow manufacturer-specific recommendations, which may vary between products

• Working solution preparation:

  • Dilute antibodies in appropriate buffers (PBS or TBS with 0.1% BSA or similar carrier protein)

  • For Western blotting applications, prepare fresh working solutions at 0.1-2.0 μg/mL concentration

  • For IHC applications, dilute to recommended concentrations (e.g., 15 μg/mL for AF235)

  • Use sterile techniques when preparing solutions to prevent microbial contamination

• Stability considerations:

  • Working dilutions are typically stable for 1-2 weeks at 4°C

  • Add preservatives like sodium azide (0.02%) to working solutions for extended storage

  • Monitor for signs of antibody degradation (loss of activity, increased background)

  • Record lot numbers and prepare new working solutions when performance decreases

• Handling precautions:

  • Avoid contamination with microorganisms or chemicals

  • Minimize exposure to extreme pH conditions

  • Avoid detergents except when specifically recommended in protocols

  • Centrifuge antibody vials briefly before opening to collect liquid at the bottom

• Quality control practices:

  • Periodically validate antibody performance using positive controls

  • Document antibody performance over time to identify degradation

  • Reserve a portion of high-performing antibody lots for critical experiments

  • Consider including internal standards in experiments to normalize for antibody performance variations

How should I interpret FGF4 expression patterns in different tissue contexts?

Interpreting FGF4 expression patterns requires careful consideration of biological context and technical factors:

• Normal tissue expression patterns:

  • FGF4 is normally expressed in adult mouse testis, intestine, and brain

  • Expression during development is dynamic and stage-specific, particularly in limb buds and cardiac valves

  • Low or undetectable expression in many adult tissues is normal and should not be interpreted as technical failure

• Cancer tissue interpretation:

  • FGF4 is expressed in several highly vascularized tumors

  • Quantitative comparison with matched normal tissues is essential for meaningful interpretation

  • Consider correlation with other angiogenic markers when studying FGF4 in cancer contexts

  • Heterogeneous expression within tumors may have biological significance

• Subcellular localization considerations:

  • FGF4 can be detected in different cellular compartments (cytoplasmic, secreted, nuclear)

  • Use appropriate controls and markers to confirm subcellular localization

  • Co-localization with FGF receptors or downstream signaling components may provide functional insights

• Comparative analysis framework:

  • Normalize expression data appropriately across samples

  • Consider both intensity of staining and percentage of positive cells

  • Use semi-quantitative scoring systems when appropriate for IHC analysis

  • Correlate protein expression (antibody-based) with mRNA expression when possible

• Technical vs. biological variation:

  • Distinguish between technical artifacts and true biological variation

  • Use multiple detection methods when possible (e.g., WB and IHC)

  • Consider biological replicates to confirm patterns of expression

How do different FGF4 antibodies compare in sensitivity and specificity across applications?

Different FGF4 antibodies vary in their performance characteristics across applications:

Key comparative insights:

• Application-specific performance:

  • For Western blotting, antibody 500-P158 offers the highest sensitivity at 0.1-0.2 μg/mL with detection limits of 1.5-3.0 ng/lane

  • For IHC applications, AF235 has been well-validated at 15 μg/mL in human breast cancer tissue

  • For neutralization assays, AF235 demonstrates functional inhibition with an ND₅₀ of 0.3-0.9 μg/mL

• Epitope-dependent characteristics:

  • Middle region antibodies (ABIN3043756) may offer better cross-species reactivity due to targeting conserved functional domains

  • Full-length antibodies may provide better sensitivity for applications like neutralization assays

• Species-specific considerations:

  • Most antibodies work well with human samples

  • For mouse or rat studies, specifically validated antibodies like ABIN3043756 or ab106355 are preferred

  • Cross-reactivity predictions based on sequence homology should be experimentally validated

• Validation evidence quality:

  • Some antibodies have more extensive validation data (specific bands in WB, clear IHC images)

  • Consider the extent and quality of validation when selecting antibodies for critical experiments

What are the emerging applications of FGF4 antibodies in advanced research techniques?

FGF4 antibodies are finding utility in several cutting-edge research applications beyond traditional techniques:

• Single-cell analysis applications:

  • Mass cytometry (CyTOF) incorporating FGF4 antibodies can profile signaling at the single-cell level

  • Single-cell Western blotting can detect FGF4 expression heterogeneity within populations

  • Imaging mass cytometry can map FGF4 distribution in tissue contexts with subcellular resolution

• Advanced imaging applications:

  • Super-resolution microscopy with FGF4 antibodies can reveal detailed subcellular localization

  • Multiplexed immunofluorescence can simultaneously detect FGF4 alongside receptors and downstream effectors

  • Intravital imaging using labeled FGF4 antibodies can track dynamics in live animal models

• Functional genomics integration:

  • ChIP-seq using antibodies against transcription factors regulated by FGF4 signaling

  • Combination of CRISPR screens with FGF4 antibody-based detection to identify pathway components

  • Spatial transcriptomics combined with FGF4 immunodetection to correlate protein and RNA localization

• Therapeutic development applications:

  • Development of neutralizing antibodies as potential therapeutic agents

  • Use of FGF4 antibodies in antibody-drug conjugates targeting FGF4-expressing tumors

  • Antibody-based imaging agents for detecting FGF4-expressing tumors in vivo

• Synthetic biology applications:

  • Engineered cellular circuits using anti-FGF4 single-chain antibodies as intracellular sensors

  • Optogenetic control of FGF4 signaling validated using antibody-based detection

  • Creation of synthetic receptors monitored via antibody-based detection of pathway activation