ZFYVE26 Antibody

What is ZFYVE26 and why is it significant in neurological research?

ZFYVE26 encodes a protein containing a FYVE zinc finger binding domain that targets proteins to membrane lipids through interaction with phospholipids. This protein is critical in neurodegenerative research because mutations in the ZFYVE26 gene are associated with autosomal recessive spastic paraplegia-15 (SPG15), a form of hereditary spastic paraplegia with thinning of the corpus callosum (ARHSP-TCC) .

ZFYVE26 is widely expressed in neurons and has been shown to co-localize with endoplasmic reticulum and endosome markers, suggesting a significant role in intracellular trafficking . Mouse models lacking Zfyve26 develop progressive spastic gait disorders closely resembling human SPG15 disease, exhibiting neurodegeneration in both motor cortex neurons and cerebellar Purkinje cells . The accumulation of autofluorescent material in lysosomal structures of Zfyve26-deficient neurons further indicates its crucial role in endolysosomal pathway trafficking .

What experimental applications are most suitable for ZFYVE26 antibodies?

ZFYVE26 antibodies can be effectively utilized in multiple experimental applications:

For optimal results, researchers should note that some commercially available antibodies directed against the C-terminus have shown better sensitivity (1:350 dilution) compared to those against the N-terminus (1:100 dilution) . Additionally, since multiple isoforms of ZFYVE26 are known to exist, antibody selection should be informed by the specific research question .

How should ZFYVE26 antibodies be properly validated before experimental use?

Proper validation of ZFYVE26 antibodies is crucial due to the protein's high molecular weight and potential cross-reactivity. A comprehensive validation approach should include:

• Knockout/knockdown controls: Testing the antibody in tissues/cells lacking ZFYVE26, such as knockout mouse models. Complete absence of the 285 kDa band confirms specificity .

• Isoform verification: Confirm detection of relevant isoforms using antibodies targeting different epitopes, as demonstrated with both N-terminal and C-terminal antibodies in published studies .

• Cross-species reactivity: Verify antibody performance across species of interest. For example, some commercial antibodies react with human, mouse, and rat ZFYVE26 .

• Positive control tissues: Include brain, liver, lung, and kidney tissues, which have demonstrated reliable ZFYVE26 expression .

• Epitope specificity: Confirm the specific region recognized by the antibody. For instance, some antibodies target amino acids 1710-1760 of human ZFYVE26 .

For definitive validation, researchers should replicate the absence of ZFYVE26-specific bands in knockout versus wild-type tissues, as demonstrated in published studies using both N-terminal and C-terminal targeted antibodies .

How can ZFYVE26 antibodies be used to study endolysosomal trafficking defects in neurodegenerative disorders?

ZFYVE26 antibodies provide valuable tools for investigating endolysosomal trafficking defects in neurodegenerative disorders through several advanced methodological approaches:

• Co-localization studies: ZFYVE26 antibodies can be combined with markers for early endosomes (EEA1) to assess protein targeting and trafficking dynamics . This approach can reveal altered distribution patterns in disease models.

• Lysosomal accumulation investigation: In Zfyve26 knockout models, autofluorescent material accumulates in lysosomes, which can be co-stained with Lamp1 and ZFYVE26 antibodies to analyze trafficking defects . Researchers should employ:

  • Multi-channel confocal microscopy with standardized acquisition settings

  • Quantitative colocalization analysis with Manders' or Pearson's coefficients

  • Time-course studies to track progression of accumulation

• Subcellular fractionation: ZFYVE26 antibodies can be used to detect the protein in various cellular fractions to determine its association with membrane compartments. This helps establish which trafficking steps are disrupted in disease states.

• AP5 complex interaction studies: Since ZFYVE26 associates with the adaptor protein 5 (AP5) complex, co-immunoprecipitation with ZFYVE26 antibodies followed by detection of AP5 components can reveal alterations in this interaction in disease models .

The methodological approach should include comparison between wild-type and disease models, with careful quantification of colocalization coefficients and statistical analysis of changes in distribution patterns across multiple biological replicates.

What considerations are important when interpreting contradictory results between N-terminal and C-terminal ZFYVE26 antibodies?

Researchers frequently encounter discrepancies between results obtained with N-terminal versus C-terminal ZFYVE26 antibodies. These contradictions require careful methodological consideration:

• Epitope accessibility: The ZFYVE26 protein's complex tertiary structure may result in differential epitope masking depending on cellular context, fixation methods, or protein interactions. N-terminal epitopes (requiring 1:100 dilution) appear less accessible than C-terminal ones (usable at 1:350 dilution) .

• Protein processing: ZFYVE26 may undergo post-translational modifications or proteolytic processing that affect epitope availability. Researchers should:

  • Run parallel experiments with both antibody types

  • Include positive controls with full-length protein expression

  • Consider denaturing conditions that may expose hidden epitopes

• Isoform specificity: Multiple ZFYVE26 isoforms exist, and antibodies targeting different regions may detect distinct isoform subsets . When contradictory results arise:

  • Verify which isoforms are expressed in your experimental system

  • Compare detected molecular weights with predicted isoform sizes

  • Use RNA analysis to correlate protein detection with transcript expression

• Experimental validation: In cases of discrepancy, verification through orthogonal methods is essential:

  • Confirm results with knockout/knockdown controls

  • Use epitope-tagged ZFYVE26 constructs as parallel controls

  • Consider alternative detection methods like mass spectrometry

In published research, western blot analysis with both N-terminal and C-terminal antibodies confirmed the complete absence of ZFYVE26 in knockout mice, suggesting both antibody types can reliably detect the protein when methodologically optimized .

How can researchers effectively use ZFYVE26 antibodies to study neurodegeneration in SPG15 disease models?

Studying neurodegeneration in SPG15 disease models with ZFYVE26 antibodies requires systematic methodological approaches:

• Progressive pathology tracking: ZFYVE26 antibodies enable temporal analysis of protein expression and localization changes during disease progression. Researchers should:

  • Establish time-course experiments spanning pre-symptomatic to advanced stages

  • Quantify ZFYVE26 levels in specific neuronal populations like cortical motoneurons and Purkinje cells

  • Correlate protein changes with onset of autofluorescent accumulation (first detected at 2 months in mouse models)

• Cell-type specific degeneration analysis: Different neuronal populations show varying vulnerability in SPG15. Methodological approaches include:

  • Co-labeling with cell-type specific markers alongside ZFYVE26 antibodies

  • Stereological quantification of affected neurons over disease course

  • Comparison of subcellular ZFYVE26 distribution between vulnerable and resistant neurons

• Lysosomal pathology characterization: Accumulation of autofluorescent material in lysosomes is a hallmark of SPG15. Research methods should include:

  • Triple labeling with ZFYVE26, lysosomal markers (Lamp1), and Sudan Black staining

  • Electron microscopy following ZFYVE26 immunogold labeling

  • Quantification of abnormal deposit size, frequency, and distribution in subcellular compartments

• Axonal transport analysis: Zfyve26 knockout motoneurons show reduced axonal outgrowth and mitochondrial transport. Methodological considerations include:

  • Live imaging of cultured neurons with dual labeling for ZFYVE26 and transport cargoes

  • Kymograph analysis of cargo movement in wild-type versus mutant neurons

  • Correlation between ZFYVE26 expression levels and transport metrics

In published mouse studies, this approach revealed progressive accumulation of autofluorescent material beginning at 2 months of age, becoming significantly larger and more frequent by 16 months, with altered subcellular localization extending into Purkinje cell dendrites .

What are the optimal protocols for detecting endogenous ZFYVE26 in immunofluorescence studies?

Detecting endogenous ZFYVE26 by immunofluorescence presents technical challenges requiring specific methodological optimizations:

• Fixation optimization: Since both commercially available N-terminal and C-terminal antibodies have shown limitations in detecting endogenous ZFYVE26 in immunofluorescence studies , researchers should evaluate multiple fixation approaches:

  • 4% paraformaldehyde fixation (10 minutes) followed by gentle permeabilization

  • Methanol fixation (-20°C, 10 minutes) for enhanced epitope accessibility

  • Dual fixation with PFA followed by methanol for balanced structure preservation and antigenicity

• Signal amplification: Given detection challenges, employ signal enhancement methods:

  • Tyramide signal amplification (TSA) systems

  • Use of high-sensitivity detection systems (e.g., QDots, high-QE cameras)

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

• Background reduction: Optimize blocking conditions using:

  • 5-10% normal serum from the species of secondary antibody origin

  • 0.1-0.3% Triton X-100 for permeabilization

  • Addition of 1% BSA to reduce non-specific binding

• Alternative approaches: When direct IF detection remains challenging:

  • Consider epitope-tagged ZFYVE26 expression systems

  • Use in situ hybridization for transcript localization as demonstrated in published studies

  • Employ proximity ligation assays for detecting ZFYVE26 interactions with higher sensitivity

The methodological challenges in immunofluorescence detection have led researchers to supplement antibody studies with non-radioactive in situ hybridization, which revealed broad ZFYVE26 expression across brain regions including the olfactory bulb, cortical neurons, hippocampus, Purkinje cells, and spinal cord neurons .

What are the recommended troubleshooting steps for Western blot detection of ZFYVE26?

Western blot detection of ZFYVE26 presents challenges due to its high molecular weight (285 kDa). A systematic troubleshooting approach includes:

• Protein extraction optimization:

  • Use RIPA buffer with protease inhibitor cocktail for complete extraction

  • Avoid excessive heating of samples (use 70°C for 10 minutes instead of 95°C)

  • Consider specialized extraction buffers for membrane-associated proteins

• Gel electrophoresis adaptation:

  • Use low percentage (6-8%) gels or gradient gels (4-15%)

  • Extend separation time to improve resolution of high molecular weight proteins

  • Reduce sample load to prevent lane overloading and smearing

• Transfer optimization:

  • Employ wet transfer systems for high molecular weight proteins

  • Extend transfer time (overnight at 30V, 4°C)

  • Add 0.1% SDS to transfer buffer to facilitate movement of large proteins

• Detection enhancement:

  • Increase primary antibody concentration (1:350 for C-terminal, 1:100 for N-terminal antibodies)

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

  • Use high-sensitivity detection systems (ECL Prime or SuperSignal West Femto)

• Signal verification:

  • Always include positive control tissues (brain, liver, lung, kidney)

  • Use knockout/knockdown controls to confirm specificity

  • Check for proteolytic degradation by including a panel of protease inhibitors

When implementing these approaches, researchers should be able to detect the characteristic 285 kDa band that represents the full-length ZFYVE26 protein, as verified in published studies with both N-terminal and C-terminal targeting antibodies .

What methodological approaches can resolve discrepancies in ZFYVE26 molecular weight detection?

Researchers often encounter variations in the detected molecular weight of ZFYVE26. The calculated molecular weight is 285 kDa, but detection may vary. Methodological approaches to resolve these discrepancies include:

• Molecular weight standard optimization:

  • Use high-range molecular weight markers that extend beyond 250 kDa

  • Include internal reference proteins of known high molecular weight

  • Generate a standard curve to accurately calculate molecular weights

• Sample preparation variables:

  • Compare reducing vs. non-reducing conditions

  • Evaluate different detergent types and concentrations

  • Test denaturation at varying temperatures (37°C, 70°C, 95°C)

• Isoform identification:

  • Design experiments to distinguish known ZFYVE26 isoforms

  • Use antibodies targeting different epitopes to identify specific fragments

  • Compare results with transcript analysis to correlate protein patterns with expressed variants

• Post-translational modification assessment:

  • Treat samples with phosphatases, glycosidases, or other enzymes to identify modifications

  • Compare migration patterns before and after treatment

  • Consider specialized techniques like Phos-tag gels for phosphorylated proteins

• Confirmation with recombinant proteins:

  • Run purified ZFYVE26 protein alongside experimental samples

  • Express tagged ZFYVE26 constructs of defined molecular weight

  • Use domain-specific constructs to map antibody reactivity

In published studies, the observed molecular weight of ZFYVE26 in SDS-PAGE (285 kDa) aligned with the calculated molecular weight, suggesting that standardized sample preparation and electrophoresis conditions can yield consistent results .

How can researchers investigate the relationship between ZFYVE26 and the adaptor protein 5 (AP5) complex?

ZFYVE26 (Spastizin) has been implicated in association with the adaptor protein 5 (AP5) complex, which is involved in endolysosomal trafficking. To investigate this relationship methodologically:

• Co-immunoprecipitation optimization:

  • Use antibodies against ZFYVE26 to pull down the protein complex

  • Probe for AP5 components (AP5Z1, AP5B1, AP5M1, AP5S1) in the precipitate

  • Include appropriate controls (IgG control, lysates from knockout tissues)

  • Optimize detergent conditions to maintain protein interactions

• Co-fractionation analysis:

  • Perform subcellular fractionation studies

  • Analyze the distribution of ZFYVE26 and AP5 components across fractions

  • Quantify co-occurrence in specific cellular compartments

  • Compare wild-type patterns with disease models

• Proximity ligation assays:

  • Use ZFYVE26 antibodies together with antibodies against AP5 components

  • Quantify interaction signals in different subcellular compartments

  • Compare results between wild-type and disease models

  • Correlate interaction patterns with functional outcomes

• Functional cargo trafficking assays:

  • Identify potential cargo molecules trafficked by the ZFYVE26-AP5 complex

  • Track cargo movement in cells with normal versus disrupted ZFYVE26

  • Quantify trafficking rates, directionality, and targeting accuracy

  • Determine if AP5 recruitment to membranes depends on ZFYVE26

Published studies have demonstrated that Zfyve26 co-fractionates with components of the AP5 complex, supporting their functional relationship in the endolysosomal system . Further investigation using these methodological approaches can help elucidate the precise mechanisms by which ZFYVE26 contributes to AP5-mediated trafficking.

What techniques can researchers use to study the role of ZFYVE26 in axonal transport?

ZFYVE26 has been implicated in axonal transport processes, with knockout studies showing reduced axonal outgrowth and mitochondrial transport in motoneurons . To investigate this function:

• Live imaging of axonal transport:

  • Culture primary neurons from wild-type and Zfyve26 knockout models

  • Label mitochondria with fluorescent markers (MitoTracker, mito-DsRed)

  • Perform time-lapse imaging (1 frame/5 seconds for 5 minutes)

  • Analyze transport parameters:

  Transport Parameter	Wild-type	Zfyve26 KO	Statistical Significance
  Anterograde velocity	Normal baseline	Reduced	p < 0.05
  Retrograde velocity	Normal baseline	Reduced	p < 0.05
  Stationary mitochondria %	Normal baseline	Increased	p < 0.05
  Run length	Normal baseline	Reduced	p < 0.05

• Axon outgrowth assays:

  • Culture embryonic motoneurons from both genotypes

  • Identify axons using Tau immunoreactivity

  • Measure axon length after 4 days in culture

  • Quantify branching patterns and complexity

• Motor protein interaction studies:

  • Investigate ZFYVE26 interaction with kinesin and dynein motor complexes

  • Perform co-immunoprecipitation with ZFYVE26 antibodies

  • Probe for motor proteins and adaptor molecules

  • Compare wild-type and disease model interactions

• Cargo-specific transport analysis:

  • Expand beyond mitochondria to examine multiple cargo types (synaptic vesicles, endosomes, lysosomes)

  • Use dual-color imaging to correlate ZFYVE26 localization with cargo movement

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to measure transport dynamics

Published studies demonstrated that axonal outgrowth was significantly reduced in Zfyve26 knockout motoneurons compared to wild-type, as was the bidirectional axonal transport rate of mitochondria, while axonal branching remained unaffected . These methodological approaches can be further refined to elucidate the specific mechanisms by which ZFYVE26 contributes to axonal transport.

What emerging methodologies might enhance ZFYVE26 research in the future?

As research on ZFYVE26 continues to evolve, several promising methodological approaches are emerging that may address current limitations:

• Super-resolution microscopy techniques: These methods can overcome the limited resolution of conventional microscopy to reveal precise subcellular localization of ZFYVE26:

  • STED (Stimulated Emission Depletion) microscopy

  • STORM (Stochastic Optical Reconstruction Microscopy)

  • PALM (Photoactivated Localization Microscopy)

• CRISPR-based approaches:

  • Genome editing to create precise disease mutations in cellular models

  • Endogenous tagging of ZFYVE26 to overcome antibody limitations

  • CRISPRi/a systems for controlled modulation of ZFYVE26 expression

• Proximity labeling methods:

  • BioID or TurboID fusions with ZFYVE26 to identify proximal interacting proteins

  • APEX2-based approaches for temporal mapping of the ZFYVE26 interactome

  • Integration with mass spectrometry for comprehensive interaction mapping

• Advanced animal models:

  • Conditional and cell-type specific knockout models

  • Humanized mouse models carrying patient-specific mutations

  • Large animal models that better recapitulate human neuroanatomy

• Single-cell approaches:

  • Single-cell transcriptomics to identify cell-specific responses to ZFYVE26 dysfunction

  • Spatial transcriptomics to map expression changes in intact tissue context

  • Correlative light and electron microscopy for ultrastructural localization

These methodological advances promise to overcome current technical challenges in ZFYVE26 research and provide deeper insights into its role in neurological disease pathogenesis.

How can researchers integrate ZFYVE26 antibody studies with other complementary approaches?

To develop a comprehensive understanding of ZFYVE26 function and dysfunction, researchers should consider integrating antibody-based studies with complementary methodologies:

• Multi-omics integration:

  • Combine antibody-based protein detection with transcriptomics data

  • Correlate proteomics and interactomics findings with functional outcomes

  • Integrate metabolomics to understand downstream effects of ZFYVE26 dysfunction

• Patient-derived models:

  • Use ZFYVE26 antibodies to characterize protein expression in patient-derived cells

  • Compare findings between different mutation types

  • Correlate molecular findings with clinical phenotypes

• Functional rescue experiments:

  • Test various ZFYVE26 constructs for their ability to rescue knockout phenotypes

  • Identify critical domains through structure-function analysis

  • Develop targeted therapeutic approaches based on mechanistic insights

• Cross-species comparative analysis:

  • Apply ZFYVE26 antibodies across model organisms

  • Identify evolutionarily conserved and divergent aspects of function

  • Leverage model-specific advantages for mechanistic studies

• Translational biomarker development:

  • Explore whether ZFYVE26 or its interacting partners could serve as biomarkers

  • Develop sensitive assays using well-validated antibodies

  • Correlate biomarker changes with disease progression

By integrating these approaches, researchers can overcome the limitations of any single methodology and develop a more comprehensive understanding of ZFYVE26's role in health and disease.