FH14 Antibody

What is FGF14 and why is it important in neurological research?

FGF14 (Fibroblast Growth Factor 14), also known as FHF4 (Fibroblast Growth Factor Homologous Factor 4), is encoded by the FGF14 gene and belongs to the fibroblast growth factor homologous factors family. Unlike traditional FGFs, FGF14 functions primarily as an intracellular modulator rather than a secreted growth factor. Its importance in neurological research stems from its critical role in regulating voltage-gated sodium channels (Nav channels), which are essential for neuronal excitability and action potential generation. FGF14 is prominently expressed in both the hippocampus and the cerebellum, where it localizes specifically to the axon initial segment. This specialized localization pattern makes it a valuable marker for studying neuronal polarity and excitability. Importantly, mutations in FGF14 are associated with Spinocerebellar Ataxia and Autosomal Dominant Cerebellar Ataxia, making it a significant target for understanding neurodegenerative disorders .

How should researchers validate the specificity of anti-FGF14 antibodies in experimental systems?

Validating antibody specificity is critical for experimental reliability. For FGF14 antibodies, implement a multi-faceted approach: First, conduct Western blot analysis with positive controls (tissues known to express FGF14, such as cerebellum) and negative controls (tissues lacking FGF14 expression or FGF14 knockout samples if available). Verify that the antibody detects a band at the expected molecular weight of approximately 28 kDa. Second, perform immunofluorescence studies comparing staining patterns in wild-type versus FGF14-knockout tissues if available. Third, pre-absorb the antibody with recombinant FGF14 protein to confirm signal elimination. Fourth, test cross-reactivity with other FGF family members, particularly closely related FHFs. The FGF14 antibody (N56/21) has been specifically tested to cross-react with both FGF14a and FGF14b isoforms but does not cross-react with FGF11, FGF12, or FGF13, demonstrating its specificity within the FHF subfamily . Each new lot of antibody should be quality control tested by immunohistochemistry on rat or mouse brain to confirm the expected staining pattern matches the established axon initial segment localization.

What are the optimal sample preparation protocols for detecting FGF14 in different experimental contexts?

Sample preparation for FGF14 detection varies by application. For immunohistochemistry and immunocytochemistry, fixation with 4% paraformaldehyde for 15-20 minutes preserves FGF14 epitopes while maintaining tissue architecture. Excessive fixation may mask epitopes, so titration of fixation time is recommended. For tissue sections, antigen retrieval using citrate buffer (pH 6.0) at 95°C for 15-20 minutes improves detection of FGF14. When preparing samples for Western blotting, use RIPA buffer supplemented with protease inhibitors, but avoid excessive heating of samples as FGF14 can be heat-sensitive. For co-immunoprecipitation studies, milder lysis conditions using NP-40 buffer better preserve protein-protein interactions. For all applications, fresh samples yield superior results compared to frozen archived materials. When working with cultured neurons, allow sufficient time (14-21 days in vitro) for FGF14 localization to the axon initial segment to be fully established, as premature analysis may yield inconsistent results .

What are the recommended dilutions and incubation conditions for FGF14 antibody across different applications?

Optimal dilutions and conditions for the anti-FGF14 antibody (N56/21) vary by application to balance signal intensity with background. For Western blotting, a 1:1000 dilution in 5% non-fat dry milk or BSA in TBST with overnight incubation at 4°C yields optimal results. For immunohistochemistry and immunocytochemistry, use a 1:500 dilution with incubation for 24-48 hours at 4°C to maximize signal-to-noise ratio. Extended incubation times at lower temperatures generally improve specific labeling. For immunoprecipitation, use 2-5 μg of antibody per 500 μg of total protein lysate. When performing double or triple immunolabeling, titrate the FGF14 antibody concentration to accommodate all primary antibodies used, typically starting with a slightly more dilute preparation (1:750). For flow cytometry applications, a 1:200 dilution is recommended. Always include a concentration gradient in pilot experiments to determine optimal conditions for your specific sample type, as tissue fixation methods and protein expression levels can necessitate adjustments to these baseline recommendations .

How should FGF14 antibody be stored to maintain long-term activity and performance?

To maintain optimal activity of the FGF14 antibody, proper storage is essential. For long-term storage, aliquot the antibody and store at ≤ -20°C, avoiding repeated freeze-thaw cycles which can cause protein denaturation and reduce antibody efficacy. For short-term storage (≤1 month), the antibody can be stored at 2-8°C. Before each use, centrifuge the vial briefly to collect all liquid at the bottom and ensure accurate concentration. The FGF14 antibody is typically supplied in a storage buffer containing 10 mM Tris, 50 mM Sodium Chloride, and 0.065% Sodium Azide at pH 7.23, which helps maintain stability. Working dilutions should be prepared fresh for each experiment rather than stored for future use. The shelf life of the antibody is approximately 24 months from the date of receipt when stored properly. If precipitation occurs, centrifuge the solution and use the supernatant. For maximum protein recovery, always centrifuge the vial prior to removing the cap to collect all material that may be trapped in the cap .

How can researchers optimize co-localization studies of FGF14 with voltage-gated sodium channels?

Optimizing co-localization studies between FGF14 and voltage-gated sodium channels (Nav) requires careful consideration of several technical parameters. First, select antibodies raised in different host species (e.g., mouse anti-FGF14 and rabbit anti-Nav) to allow simultaneous detection without cross-reactivity. Second, implement sequential staining protocols in cases where available antibodies are from the same species—complete the first immunolabeling, fix with 4% paraformaldehyde, and then proceed with the second antibody. Third, use super-resolution microscopy techniques such as STED or STORM rather than conventional confocal microscopy to accurately resolve the nanoscale co-localization at the axon initial segment, where both proteins are concentrated in close proximity. Fourth, employ rigorous quantitative co-localization analysis using Pearson's or Mander's coefficients rather than relying on visual assessment alone. Fifth, include appropriate controls including single-labeled samples to establish bleed-through thresholds and competitive peptide blocking to confirm specificity. Given that FGF14 physically interacts with Nav channels to modulate their function, proximity ligation assays (PLA) can provide additional confirmation of direct protein-protein interactions beyond co-localization .

What methodological considerations are important when studying FGF14 in models of Spinocerebellar Ataxia?

When studying FGF14 in Spinocerebellar Ataxia (SCA) models, several methodological considerations are critical for meaningful results. First, carefully select the appropriate animal model that recapitulates the specific SCA pathology of interest, noting that FGF14 is particularly associated with SCA27. Second, establish appropriate timepoints for analysis, as FGF14 abnormalities may precede clinical manifestations—typically examining pre-symptomatic, early symptomatic, and late disease stages. Third, implement comprehensive behavioral testing (rotarod, beam walking, gait analysis) to correlate molecular findings with functional outcomes. Fourth, use quantitative immunohistochemistry to assess not only FGF14 expression levels but also its subcellular localization, as mislocalization may occur before expression changes. Fifth, combine protein analysis with electrophysiological recordings to determine functional consequences of FGF14 alterations on neuronal excitability. Sixth, employ Purkinje cell-specific manipulations to distinguish cell-autonomous effects from secondary consequences. Finally, when analyzing human samples, account for post-mortem interval effects on FGF14 detection, as the protein may undergo degradation or relocalization after death .

How can researchers troubleshoot false negative results when detecting FGF14 in brain tissue sections?

False negative results when detecting FGF14 in brain tissue sections can stem from multiple factors requiring systematic troubleshooting. First, verify antibody viability through positive control experiments using cerebellum or hippocampus tissue where FGF14 is abundantly expressed. Second, optimize antigen retrieval methods—FGF14 epitopes are particularly sensitive to fixation artifacts, so test multiple retrieval methods (heat-induced epitope retrieval with citrate buffer pH 6.0, Tris-EDTA pH 9.0, or enzymatic retrieval with proteinase K). Third, extend primary antibody incubation time to 48-72 hours at 4°C to enhance penetration into tissue. Fourth, reduce section thickness to 20-30 μm if using free-floating sections to improve antibody penetration. Fifth, test signal amplification systems such as tyramide signal amplification or polymer-based detection systems that can significantly enhance detection sensitivity. Sixth, check for interfering factors in your experimental system—high lipid content in brain tissue can impede antibody penetration, so additional permeabilization with 0.3% Triton X-100 may help. Finally, consider that expression levels naturally vary between brain regions and developmental stages, so adjust experimental design accordingly .

What are the critical parameters for quantifying changes in FGF14 expression in disease models using immunoblotting?

Quantifying FGF14 expression changes in disease models via immunoblotting requires attention to several critical parameters. First, standardize tissue sampling to ensure consistent anatomical regions are compared, as FGF14 expression varies significantly between brain areas. Second, optimize protein extraction—use RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors, especially when studying post-translational modifications of FGF14. Third, validate protein loading with multiple housekeeping controls (β-actin, GAPDH) to account for potential disease-related changes in traditional reference proteins. Fourth, determine the linear dynamic range of detection for your specific antibody lot and ensure samples fall within this range—FGF14 antibody typically works well at 1:1000 dilution for Western blotting. Fifth, implement technical replicates (minimum triplicate) and biological replicates (n≥5) to account for variability. Sixth, quantify both the 28 kDa main band and any additional bands that may represent alternatively spliced isoforms or post-translationally modified forms of FGF14. Finally, normalize results to total protein content using stain-free technology or Ponceau staining rather than relying solely on housekeeping proteins, which can be altered in many disease states .

How does epitope accessibility impact the detection of FGF14 in different experimental contexts?

Epitope accessibility significantly impacts FGF14 detection across different experimental contexts, requiring tailored approaches for optimal results. The anti-FGF14 antibody (N56/21) recognizes a fusion protein containing amino acids 1-252 of mouse FGF14b, but accessibility of these epitopes varies contextually. In fixed tissues, aldehyde crosslinking can mask epitopes, necessitating optimization of antigen retrieval methods—typically heat-induced epitope retrieval with citrate buffer (pH 6.0) for 15-20 minutes. For Western blotting, denaturing conditions fully expose linear epitopes, making detection straightforward, but native gel electrophoresis may yield weaker signals as conformational changes can hide binding sites. In immunoprecipitation studies, the antibody performs well because it recognizes both native and partially denatured forms of FGF14. When FGF14 participates in protein complexes, particularly with voltage-gated sodium channels at the axon initial segment, steric hindrance can limit antibody access, requiring more stringent permeabilization with 0.3% Triton X-100 rather than the milder 0.1% typically used. For dual-labeling experiments, consider the order of antibody application, as large antibody complexes from the first staining round can block access to nearby epitopes in subsequent rounds .

What considerations are important when developing quantitative imaging workflows for FGF14 at the axon initial segment?

Developing quantitative imaging workflows for FGF14 at the axon initial segment (AIS) requires specialized approaches to capture this spatially restricted signal accurately. First, implement z-stack acquisition with optimal step size (0.3-0.5 μm) to fully capture the three-dimensional structure of the AIS. Second, use appropriate co-markers such as AnkyrinG or βIV-spectrin to definitively identify the AIS compartment. Third, establish standardized intensity thresholds for AIS identification based on these co-markers before quantifying FGF14 signal. Fourth, measure multiple parameters beyond just intensity—include AIS length, distance from soma, and intensity profile along the AIS, as FGF14 distribution is not uniform and changes in pattern rather than total amount may be biologically significant. Fifth, employ deconvolution algorithms to improve signal resolution, but apply consistently across all samples. Sixth, implement automated analysis pipelines using ImageJ/FIJI with plugins such as Simple Neurite Tracer or NeuronJ to reduce investigator bias. Finally, report detailed microscopy parameters (objective numerical aperture, pinhole size, laser power, gain settings) to ensure reproducibility, as small variations in image acquisition parameters can dramatically affect quantitative measurements of this spatially restricted protein .

How can researchers distinguish between FGF14a and FGF14b isoforms in experimental systems?

Distinguishing between FGF14a and FGF14b isoforms presents a significant challenge as these splice variants differ only in their N-terminal regions but share identical C-terminal domains. The commercially available anti-FGF14 antibody (N56/21) recognizes both isoforms, making direct immunological distinction difficult. To overcome this limitation, researchers should employ a multi-modal approach: First, use RT-qPCR with isoform-specific primers targeting the unique N-terminal coding regions to quantify relative mRNA expression of each variant. Second, perform Western blotting with high-resolution gel systems (10-12% polyacrylamide) that can potentially separate the slightly different molecular weights (FGF14a: 27 kDa; FGF14b: 28 kDa). Third, when studying cellular localization, use expression constructs with isoform-specific tags (e.g., FGF14a-GFP and FGF14b-mCherry) for direct visualization of differential distribution. Fourth, employ functional assays to distinguish isoforms based on their different effects on sodium channel gating—FGF14b typically causing more pronounced shifts in activation curves compared to FGF14a. Finally, for absolute confirmation, mass spectrometry can identify isoform-specific peptides from immunoprecipitated samples .

What methodological approaches are recommended for studying FGF14 interactions with voltage-gated ion channels?

Studying FGF14 interactions with voltage-gated ion channels requires specialized methodologies that preserve native protein-protein interactions while providing quantitative data. First, co-immunoprecipitation using anti-FGF14 antibody can pull down associated channel proteins—use mild lysis conditions (1% NP-40 or 1% digitonin rather than harsher RIPA buffer) to preserve complexes. Second, proximity ligation assay (PLA) offers in situ visualization of FGF14-channel interactions with nanometer resolution—this technique is particularly valuable for examining interaction changes under different physiological or pathological conditions. Third, fluorescence resonance energy transfer (FRET) using tagged constructs provides dynamic information about protein interactions in living cells—typical pairings include FGF14-CFP with YFP-tagged channel subunits. Fourth, surface plasmon resonance with recombinant proteins quantifies binding kinetics and affinities—important for comparing how disease-associated mutations affect interaction strength. Fifth, patch-clamp electrophysiology combined with acute molecular interventions (peptide competitors or dominant-negative constructs) can correlate biochemical interactions with functional outcomes. Finally, bimolecular fluorescence complementation (BiFC) offers another approach where fragments of fluorescent proteins fused to FGF14 and channel proteins reconstitute fluorescence only when the proteins interact .

How does epitope mapping influence the selection of FGF14 antibody for different research applications?

Epitope mapping significantly influences FGF14 antibody selection based on the specific research application. Understanding the recognized epitope's properties is crucial for experimental success. The anti-FGF14 antibody (N56/21) recognizes a fusion protein containing amino acids 1-252 of mouse FGF14b, making it suitable for most standard applications. For immunohistochemistry applications, this epitope location enables robust detection at the axon initial segment where FGF14 naturally concentrates. When studying protein-protein interactions, consider whether the antibody's epitope overlaps with known binding sites for voltage-gated sodium channels or other interaction partners. Based on studies of related proteins like Factor H, where precise epitope mapping has been conducted, we know that single amino acid changes can dramatically alter antibody binding affinity, with reported decreases in binding observed with mutations at key positions . For functional blocking experiments, select antibodies whose epitopes directly overlap with functional domains. When developing sandwich ELISA assays, pairs of antibodies recognizing non-overlapping epitopes are essential. For detection of post-translationally modified FGF14, ensure the antibody's epitope doesn't contain potential modification sites that could mask recognition .

Table 1: Technical Specifications of Anti-FGF14/FHF4 Antibody

Table 2: Recommended Dilutions for Different Applications

Table 3: Comparison of Patient FH Autoantibody Levels Before and After Treatment

This data demonstrates the significant reduction in autoantibody levels following treatment and provides important benchmarks for researchers studying antibody-mediated conditions .