FGF13 Antibody

How do I select the appropriate FGF13 antibody for my research applications?

Selecting the optimal FGF13 antibody requires consideration of multiple experimental parameters:

• Target specificity: Determine which FGF13 isoform(s) you need to detect. Some antibodies (e.g., those targeting aa 2-18) detect specific isoforms like FGF13A/FHF2A but may cross-react with other FHF family members (FGF11A/FHF3A, FGF12A/FHF1A, FGF14A/FHF4A) . For pan-FGF13 detection, select antibodies targeting conserved regions present in all variants.

• Application compatibility: Verify the antibody has been validated for your specific application:

  • Western blot (WB): Most FGF13 antibodies detect a band at ~28-30 kDa

  • Immunocytochemistry/Immunofluorescence (ICC/IF)

  • Immunohistochemistry (IHC)

• Species reactivity: Confirm reactivity with your experimental model. Many FGF13 antibodies react with human, mouse, and rat samples, but verification is essential .

• Clonality consideration:

  • Monoclonal antibodies (e.g., clone S235-22) offer high specificity and reproducibility

  • Polyclonal antibodies may provide greater sensitivity but potential batch variation

• Conjugation needs: Available options include unconjugated, FITC, HRP, or AP-conjugated antibodies, depending on your detection system .

Always validate antibodies in your specific experimental system before proceeding with full experiments.

What are the recommended validation experiments for a new FGF13 antibody?

Thorough validation ensures reliable results and should include:

• Positive control testing: Use tissues/cells known to express FGF13 (e.g., brain tissue, neuroblastoma cell lines SK-N-BE or SH-SY5Y) .

• Western blot analysis: Confirm detection of the expected molecular weight band (~28 kDa) with proper positive controls. Important validation points include:

  • Using reducing conditions with DTT or β-mercaptoethanol

  • Including molecular weight markers

  • Testing various antibody concentrations (typically 1:200-1:1000 dilution range)

• Specificity verification:

  • RNA interference: Compare signal between FGF13 knockdown and control samples

  • Overexpression systems: Test in FGF13-transfected versus mock-transfected cells

  • Peptide competition: Pre-incubate antibody with immunizing peptide to block specific binding

• Cross-reactivity assessment:

  • Test against other FHF family members (FGF11, FGF12, FGF14)

  • Evaluate potential cross-reactivity with other FGF family proteins

• Application-specific validation:

  • For IF/ICC: Include appropriate negative controls (omitting primary antibody)

  • For IHC: Test different antigen retrieval methods (citrate buffer pH 6.0 vs. TE buffer pH 9.0)

Document all validation steps thoroughly with images and experimental conditions for reproducibility.

What are the optimal conditions for detecting FGF13 by Western blot?

Successful FGF13 Western blot detection requires careful optimization:

• Sample preparation:

  • For tissue samples: Use membrane-enriched fractions for better detection

  • For cells: Acid-ethanol fixation improves FGF13 detection in some contexts

  • Include protease inhibitors to prevent degradation

• Protein loading: 15-20 μg of total protein per lane is typically sufficient

• Separation conditions:

  • 10-12% SDS-PAGE gels provide optimal resolution for the 28 kDa FGF13 protein

  • Run time: Approximately 60-90 minutes at 100V

• Transfer parameters:

  • Semi-dry or wet transfer (wet transfer generally preferred)

  • Transfer for 60-90 minutes at 100V (or overnight at 30V)

  • PVDF membranes often yield better results than nitrocellulose for FGF13

• Blocking and antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST (1 hour at room temperature)

  • Primary antibody dilutions:

    • Monoclonal antibodies: 1:200-1:500

    • Polyclonal antibodies: 1:500-1:1000

  • Incubate primary antibody overnight at 4°C

  • Secondary antibody incubation: 1 hour at room temperature

• Detection method:

  • ECL detection works well, with exposure times of approximately 1-6 minutes

  • For low expression, enhanced chemiluminescence substrates or longer exposure may be necessary

• Expected results:

  • Main band at approximately 28 kDa

  • Potential secondary bands may represent different isoforms or post-translational modifications

If signal is weak, consider membrane extraction protocols to concentrate the protein of interest.

How should I optimize immunofluorescence protocols for FGF13 detection in different neural tissues?

Optimizing IF/ICC protocols for FGF13 requires attention to fixation and permeabilization steps:

• Fixation options:

  • 4% paraformaldehyde (15 minutes at room temperature) works well for most neural tissues

  • Acid-ethanol fixation (30 minutes) has been reported to improve detection in some contexts

  • Avoid methanol fixation which can disrupt certain epitopes

• Permeabilization:

  • 0.1-0.3% Triton X-100 in PBS (10 minutes) for sufficient access to intracellular targets

  • For nuclear FGF13 detection, ensure adequate permeabilization time

• Blocking conditions:

  • 5-10% normal serum (species of secondary antibody) with 1% BSA in PBS

  • Block for 1 hour at room temperature

• Antibody concentrations:

  • Primary antibody: 1:50-1:100 dilution for most FGF13 antibodies

  • Secondary antibody: 1:500-1:1000 for fluorophore-conjugated secondaries

• Incubation times:

  • Primary antibody: Overnight at 4°C or 1-2 hours at room temperature

  • Secondary antibody: 1 hour at room temperature

• Special considerations for neural tissues:

  • For brain slices: Use thinner sections (10-20 μm) and longer antibody incubation

  • For cultured neurons: Fix at different developmental stages to track expression changes

  • For neuroblastoma cell lines: Use as positive controls (SK-N-BE or SH-SY5Y)

• Multi-label experiments:

  • Combine with subcellular markers:

    • Nuclear: DAPI or Hoechst

    • Cytoskeletal: Phalloidin (F-actin), tubulin antibodies

    • Neuronal: Map2, β-III-tubulin

• Visualization parameters:

  • Confocal microscopy recommended for precise subcellular localization

  • Z-stack imaging to fully capture distribution in complex neural tissues

Subcellular localization can vary with physiological state, particularly nuclear translocation during hypertrophy or stress conditions .

How can I distinguish between different FGF13 isoforms in my experiments?

FGF13 has multiple isoforms (FGF13-S, FGF13-U, FGF13-V, FGF13-Y, FGF13-VY) with distinct subcellular localizations and functions. To distinguish between them:

• Antibody selection strategies:

  • N-terminal targeting antibodies: Detect specific isoforms (e.g., antibodies against aa 2-18 detect FGF13A/FHF2A but not FGF13B/FHF2B)

  • C-terminal targeting antibodies: Detect multiple or all isoforms (pan-FGF13)

  • Verify epitope region in product documentation

• Transcript-level analysis:

  • Design isoform-specific primers for qRT-PCR/sqRT-PCR

  • Example approach: Researchers have quantified relative expression of five FGF13 isoforms in cardiomyocytes using isoform-specific primers

  • RNA-seq analysis with isoform-specific alignment

• Protein-level identification:

  • 2D gel electrophoresis followed by western blotting

  • Immunoprecipitation with isoform-specific antibodies

  • Mass spectrometry for definitive isoform identification

• Subcellular localization discrimination:

  • FGF13-S (FGF13A/FHF2A): Predominantly nuclear localization

  • FGF13B/FHF2B: Primarily cytoplasmic and membrane-associated

  • Use subcellular fractionation combined with western blotting

• Expression vectors for controls:

  • Create isoform-specific expression constructs as positive controls

  • Transfect cells with individual isoforms for antibody validation

  • Example: Human FGF13 cDNA variants 1, 2, and 3/5 have been subcloned into pcDNA3.1 for expression studies

The relative abundance of isoforms varies by tissue and condition. For instance, FGF13-S mRNA levels are significantly higher in cardiomyocytes isolated from TAC-surgery mouse hearts compared to sham-surgery controls .

What methods can detect changes in FGF13 subcellular localization during physiological stress?

FGF13 undergoes dynamic subcellular redistribution, particularly nuclear accumulation during stress conditions. To detect these changes:

• Subcellular fractionation protocols:

  • Separate nuclear, cytoplasmic, and membrane fractions using differential centrifugation

  • Verify fraction purity with compartment-specific markers:

    • Nuclear: Lamin A/C, Histone H3

    • Cytoplasmic: GAPDH, α-tubulin

    • Membrane: Na+/K+ ATPase, Caveolin-1

  • Quantify relative FGF13 levels in each fraction by western blot

• Live-cell imaging approaches:

  • Generate FGF13-GFP fusion constructs for real-time tracking

  • Perform time-lapse microscopy during stress induction

  • Quantify nuclear/cytoplasmic signal ratio changes

• Fixed-cell immunofluorescence analysis:

  • Co-stain with nuclear markers (DAPI, Hoechst)

  • Collect samples at different time points after stress induction

  • Quantify nuclear:cytoplasmic signal ratio

• Stress models for inducing translocation:

  • Pressure overload: TAC surgery in mice

  • Hypertrophic stimulus: Isoproterenol (ISO) treatment of cardiomyocytes

  • Cancer drug resistance: Cisplatin exposure

  • These models have demonstrated FGF13 nuclear accumulation in stressed cells

• Quantification methods:

  • Image analysis software (ImageJ/FIJI) with nuclear mask creation

  • High-content imaging systems for population-level analysis

  • Flow cytometry with permeabilized cells and fluorescent antibodies

Research has shown that FGF13 is predominantly localized in the cytoplasm in normal conditions but shows remarkable nuclear accumulation in TAC surgery mouse hearts and ISO-treated cardiomyocytes .

How can I assess the interaction between FGF13 and voltage-gated sodium channels using antibody-based approaches?

Investigating FGF13-sodium channel interactions requires specialized techniques:

• Co-immunoprecipitation (Co-IP) protocols:

  • Immunoprecipitate with anti-FGF13 antibody and probe for sodium channel subunits

  • Reverse approach: IP with anti-sodium channel antibodies and detect FGF13

  • Use mild detergents (0.5-1% Triton X-100) to preserve protein-protein interactions

  • Include controls: IgG control, input lysate, unbound fraction

• Proximity ligation assay (PLA):

  • Allows visualization of protein interactions in situ

  • Requires primary antibodies from different species:

    • Mouse anti-FGF13

    • Rabbit anti-sodium channel subunit

  • PLA signal appears as distinct puncta where proteins are in close proximity (<40 nm)

• FRET/BRET analysis:

  • Generate fluorescent protein fusions (FGF13-CFP and Nav-YFP)

  • Measure energy transfer as indicator of direct interaction

  • Calculate FRET efficiency to estimate interaction strength

• Immunofluorescence co-localization:

  • Double-label immunostaining:

    • Anti-FGF13 (e.g., mouse monoclonal)

    • Anti-sodium channel (different species antibody)

  • Analyze using confocal microscopy with co-localization plugins

  • Calculate Pearson's correlation coefficient for quantification

• Surface plasmon resonance (SPR):

  • Immobilize purified sodium channel components

  • Flow purified FGF13 (detected with anti-FGF13)

  • Measure binding kinetics and affinity constants

• Functional assays to validate interactions:

  • Patch-clamp electrophysiology in cells with altered FGF13 expression

  • Sodium current measurements at different temperatures (FGF13 regulates temperature-dependent gating)

Research has demonstrated that FGF13 regulates voltage-gated sodium channel transport and function, with deficiency in FGF13 suppressing cardiac sodium currents at elevated temperatures .

What antibody-based methods can determine if FGF13 is secreted from cells despite lacking a canonical secretion signal?

Recent research suggests FGF13 may be secreted through unconventional pathways. To investigate this phenomenon:

• Extracellular FGF13 detection strategies:

  • Collect conditioned media from cells expressing FGF13

  • Concentrate proteins by TCA precipitation or ultrafiltration

  • Perform western blot to detect FGF13 in media fractions

  • Compare levels between wild-type and FGF13-overexpressing cells

• Cell surface biotinylation assay:

  • Label cell surface proteins with membrane-impermeable biotin reagents

  • Isolate biotinylated proteins with streptavidin beads

  • Probe for FGF13 in biotinylated fraction by western blot

  • Include controls: cytoplasmic protein marker, membrane protein marker

• Secretion pathway investigation:

  • Apply classical secretion pathway inhibitors:

    • Brefeldin A (ER-Golgi transport)

    • Monensin (trans-Golgi transport)

  • Apply unconventional secretion inhibitors:

    • Methylamine (affects FGF2 secretion)

    • Ouabain (inhibits Na+/K+ ATPase)

  • Monitor effects on extracellular FGF13 levels

• Vesicular trafficking analysis:

  • Immunostain for FGF13 alongside vesicular markers:

    • Exosomal markers (CD63, CD9)

    • Secretory vesicle markers (VAMP2)

  • Isolate extracellular vesicles by ultracentrifugation

  • Probe for FGF13 in vesicular fractions

• FGFR binding studies:

  • Immobilize FGFR1-Fc fusion proteins

  • Flow conditioned media containing potential secreted FGF13

  • Detect bound FGF13 with specific antibodies

  • Compare binding affinities with known FGFR ligands

Research has shown that despite lacking a canonical signal peptide, FHFs (including FGF13) can be exported to the extracellular space through mechanisms potentially similar to the unconventional secretion of FGF2 .

How can FGF13 antibodies be used to assess its role in cancer drug resistance?

FGF13 has been implicated in platinum drug resistance in cancer. To investigate this mechanism:

• Patient sample analysis protocols:

  • Immunohistochemistry on cancer biopsies before/after treatment

  • Use validated FGF13 antibodies at optimized dilutions (1:50-1:500)

  • Score expression levels and correlation with treatment response

  • Example: In cervical cancer biopsy samples, FGF13-positive cells were more abundant in poor prognosis patients after cisplatin chemoradiotherapy

• Cell culture resistance models:

  • Compare FGF13 expression in parent vs. drug-resistant cell lines:

    • Western blot quantification (1:200-1:1000 dilution)

    • Immunofluorescence for subcellular localization

  • Knockdown/overexpression validation:

    • siRNA or shRNA targeting FGF13

    • FGF13 expression vectors (variant-specific)

    • Monitor drug sensitivity changes via viability assays

• Mechanistic studies:

  • Measure intracellular drug accumulation after FGF13 manipulation

  • Co-IP to identify FGF13 interaction partners in resistant cells

  • Investigate links between FGF13 and transporters/pumps

  • Example methodology: ICP Atomic Emission Spectrometry to measure intracellular platinum concentrations after cisplatin exposure

• Multi-parameter analysis:

  • Co-stain for FGF13 and other resistance markers

  • Quantify proliferation markers (BrdU, Ki-67) in FGF13-expressing cells

  • Analyze cell cycle distribution and apoptosis rates

Research has shown that FGF13 expression is strongly upregulated in cisplatin-resistant HeLa cells, and suppression of FGF13 expression abolished both the cells' resistance to platinum drugs and their ability to maintain low intracellular platinum levels .

What are the optimal protocols for examining FGF13's role in cardiac hypertrophy using antibody-based approaches?

FGF13 plays a role in cardiac pathophysiology, particularly hypertrophy. To investigate this function:

• Animal model tissue processing:

  • Pressure overload models (TAC surgery)

  • Fix cardiac tissue in 4% paraformaldehyde

  • Process for both paraffin sections and frozen sections

  • Optimize antigen retrieval (TE buffer pH 9.0 recommended)

• Cardiomyocyte isolation and analysis:

  • Isolate primary cardiomyocytes from normal and hypertrophic hearts

  • Immunofluorescence to track FGF13 expression and localization

  • Western blot comparison between cell types:

    • Cardiomyocytes (CMs)

    • Cardiac fibroblasts (CFs)

  • Example: FGF13 was predominantly increased in cardiomyocytes rather than cardiac fibroblasts in TAC mouse hearts

• Gain/loss-of-function studies:

  • AAV9-mediated cardiac-specific expression/knockdown

  • Validate using western blot and immunofluorescence

  • Assess cardiac function (echocardiography)

  • Measure hypertrophic markers (ANF, BNP, cell size)

  • Example: FGF13 knockdown decreased heart weight/body weight ratios and improved cardiac function in TAC mice

• Mechanistic pathway investigation:

  • Co-IP to detect FGF13-p65 interaction in heart lysates

  • Immunofluorescence for co-localization of FGF13 with NF-κB components

  • Nuclear fractionation to quantify FGF13 nuclear accumulation

  • Example: FGF13 directly interacts with p65 and co-localizes with it in the nucleus during cardiac hypertrophy

Research has shown that endogenous FGF13 is upregulated in cardiac hypertrophy with increased nuclear localization, and this upregulation plays a deteriorating role in hypertrophic cardiomyocytes and mouse hearts .

What are common technical issues with FGF13 antibodies and how can they be resolved?

Researchers often encounter specific challenges when working with FGF13 antibodies:

• Low signal intensity in western blots:

  • Problem: Weak or absent bands despite proper loading

  • Solutions:

    • Increase protein loading (20-30 μg per lane)

    • Optimize primary antibody concentration (try 1:200 instead of 1:1000)

    • Extended primary antibody incubation (overnight at 4°C)

    • Use more sensitive detection systems (ECL Prime/Femto)

    • Membrane extraction to concentrate target protein

    • Extended exposure time (up to 6 minutes has been effective)

• High background in immunofluorescence:

  • Problem: Non-specific staining obscuring specific signal

  • Solutions:

    • More extensive blocking (5% BSA + 5% normal serum, 2 hours)

    • Reduce primary antibody concentration (try 1:100 instead of 1:50)

    • Include 0.1% Tween-20 in antibody dilution buffer

    • Additional washing steps (5× 5 minutes)

    • Use specific FGF13 blocking peptide as control

• Inconsistent subcellular localization:

  • Problem: Variable nuclear/cytoplasmic distribution

  • Solutions:

    • Standardize fixation time and conditions

    • Consider cell state (FGF13 relocates during stress responses)

    • Maintain consistent culture conditions

    • Verify antibody specificity for particular isoforms

    • Control for cellular stress that might induce translocation

• Cross-reactivity with other FHF family members:

  • Problem: Antibody detects multiple FHF proteins

  • Solutions:

    • Verify epitope specificity in product documentation

    • Include appropriate knockout/knockdown controls

    • Consider using isoform-specific antibodies

    • Validate with recombinant protein standards for each FHF

• Fixation-sensitive epitopes:

  • Problem: Certain fixatives mask the FGF13 epitope

  • Solutions:

    • Compare different fixation methods (formaldehyde vs. acid-ethanol)

    • Optimize antigen retrieval (citrate pH 6.0 vs. TE pH 9.0)

    • Try epitope unmasking techniques (heat-induced vs. protease)

    • Consider live-cell staining for surface epitopes

Most FGF13 antibodies work optimally for western blot at 1:500 dilution and for immunofluorescence at 1:50-1:100 dilution .

How can I distinguish between genuine FGF13 signaling and technical artifacts in my experiments?

Distinguishing real biological effects from artifacts requires rigorous controls:

• Comprehensive antibody validation controls:

  • Positive controls:

    • Tissues known to express FGF13 (brain, neuroblastoma cell lines)

    • Recombinant FGF13 protein standards

    • FGF13-overexpressing cells

  • Negative controls:

    • FGF13 knockdown/knockout samples

    • IgG isotype controls (same species as primary)

    • Omission of primary antibody

    • Pre-absorption with immunizing peptide

• Expression manipulation verification:

  • Confirm knockdown/overexpression by multiple methods:

    • qRT-PCR for transcript levels

    • Western blot for protein levels

    • Immunofluorescence for localization changes

  • Use multiple siRNA/shRNA sequences to control for off-target effects

  • Example: Two stable HeLa cisR FGF13-knockdown clones (Kd#1 and Kd#2) with confirmed suppression at both mRNA and protein levels

• Functional assay controls:

  • Include rescue experiments:

    • Re-express FGF13 in knockdown cells

    • Use mutant versions to identify critical domains

  • Demonstrate dose-dependence of observed effects

  • Use both gain- and loss-of-function approaches

• Reproducibility considerations:

  • Test multiple antibody clones/lots

  • Validate in different cell types/tissues

  • Use alternative detection methods (e.g., mass spectrometry)

  • Perform biological replicates with statistical analysis

• Technical artifact elimination:

  • For western blots:

    • Run molecular weight markers

    • Include loading controls (GAPDH, β-actin)

    • Test different lysis buffers and conditions

  • For immunostaining:

    • Check for autofluorescence

    • Include single-stain controls in multi-label experiments

    • Test for fluorophore bleed-through

Research has shown significant consistency in FGF13 upregulation across different experimental models of cardiac hypertrophy (TAC surgery and ISO treatment), strengthening confidence in the biological relevance of these findings .

How can FGF13 antibodies be employed to investigate its potential secretion and receptor interaction?

Recent findings suggest FGF13 may be secreted and interact with FGF receptors, opening new research avenues:

• Secretion detection methodologies:

  • Conditioned media analysis:

    • Collect media from cultured cells

    • Concentrate proteins by TCA precipitation or ultrafiltration

    • Western blot with FGF13 antibodies (1:500 dilution)

    • Compare wild-type vs. FGF13-overexpressing cells

  • Pulse-chase experiments:

    • Label cells with radioactive amino acids

    • Chase with non-radioactive media

    • Immunoprecipitate FGF13 from media at multiple timepoints

    • Track appearance of labeled protein in extracellular space

• Receptor interaction studies:

  • Solid-phase binding assays:

    • Coat plates with recombinant FGFR1-4

    • Add purified FGF13 or conditioned media

    • Detect binding with anti-FGF13 antibodies

    • Measure binding affinities and compare with classical FGFs

  • Cell-based binding studies:

    • Flow cytometry with live cells expressing FGFRs

    • Add labeled FGF13 (fluorescent-tagged or antibody-detected)

    • Quantify binding with/without heparin/heparan sulfate

    • Compete with known FGFR ligands

• Signaling pathway activation detection:

  • Phospho-specific antibody panels:

    • Treat cells with purified FGF13

    • Detect activation of FGFR downstream pathways:

      • MAPK/ERK pathway

      • PI3K/AKT pathway

      • PLCγ pathway

    • Compare with classical FGF activation patterns

  • Internalization tracking:

    • Fluorescently label FGF13

    • Track receptor-mediated endocytosis by live-cell imaging

    • Co-stain with endosomal markers

    • Compare kinetics with classical FGFs

• Functional outcome measurement:

  • Anti-apoptotic response quantification:

    • Treat cells with recombinant FGF13

    • Measure apoptotic markers (Annexin V, caspase activity)

    • Block with FGFR inhibitors to confirm specificity

    • Use FGF13 antibodies for neutralization experiments

Research has shown that secreted FHFs (including FGF13) are biologically active and can trigger signaling in cells expressing FGF receptors, resulting in an anti-apoptotic response .

What methodologies can be used to investigate FGF13's role in microtubule dynamics and neuronal function?

FGF13 functions as a microtubule-binding protein affecting neuronal development. To investigate these roles:

• Microtubule interaction analysis:

  • In vitro tubulin binding assays:

    • Purify recombinant FGF13

    • Incubate with purified tubulin

    • Detect complex formation by co-sedimentation

    • Analyze binding affinity and stoichiometry

  • Microtubule polymerization assays:

    • Monitor tubulin polymerization kinetics (turbidity assay)

    • Compare rates with/without FGF13

    • Test effects of FGF13 variants/mutants

    • Examine temperature dependence of effects

• Live-cell microtubule dynamics imaging:

  • EB1-GFP tracking:

    • Transfect neurons with EB1-GFP (marks growing microtubule ends)

    • Co-express FGF13 or FGF13 shRNA

    • Track microtubule growth rate, catastrophe frequency

    • Quantify using automated tracking software

  • Photoactivation experiments:

    • Express photoactivatable tubulin-GFP

    • Activate in specific neuronal compartments

    • Track microtubule stability with/without FGF13

    • Measure fluorescence decay rates

• Neuronal morphology and migration analysis:

  • Axon/dendrite development quantification:

    • Culture primary neurons with FGF13 manipulation

    • Immunostain for axonal/dendritic markers

    • Measure branching complexity, process length

    • Analyze growth cone dynamics

    • Example: FGF13 may participate in axon refinement by negatively regulating axonal branching

  • Neuronal migration assays:

    • In utero electroporation of FGF13 constructs

    • Assess cortical neuron migration in brain slices

    • Quantify distance traveled, migration speed

    • Example: FGF13 plays a crucial role in neuron polarization and migration in the cerebral cortex and hippocampus

• Synaptic development studies:

  • GABAergic synapse analysis:

    • Immunostain for GABAergic synaptic markers

    • Co-label with FGF13 antibodies

    • Quantify synapse density in control vs. FGF13-deficient neurons

    • Example: FGF13 is required for development of axonal initial segment-targeting inhibitory GABAergic synapses by chandelier neurons