FXYD7 Antibody

What is FXYD7 and why is it significant in neuroscience research?

FXYD7 is a brain-specific member of the FXYD family of small proteins that function as tissue-specific regulators of Na,K-ATPase. Expressed exclusively in the brain, FXYD7 is a type I membrane protein with N-terminal post-translational modifications, primarily O-glycosylations on threonine residues, which are important for protein stabilization . Its significance in neuroscience lies in its isoform-specific regulation of Na,K-ATPase, particularly its ability to decrease the apparent K+ affinity of α1–β1 and α2–β1 isozymes without affecting α3–β1 isozymes . This selective modulation suggests FXYD7 plays an important role in neuronal excitability by fine-tuning ion transport across neuronal membranes.

Which applications are most suitable for FXYD7 antibodies?

Based on validated antibody products, FXYD7 antibodies are primarily suitable for the following applications:

When selecting an application, consider that FXYD7 has been most reliably detected in brain tissue samples from human, mouse, and rat models . The antibody choice should align with your specific experimental needs and the species you are investigating.

What is the optimal sample preparation protocol for detecting FXYD7 in brain tissue?

For optimal detection of FXYD7 in brain tissue, the following protocol is recommended based on published research methods:

• Tissue collection and processing: Harvest fresh brain tissue and either process immediately or flash-freeze in liquid nitrogen. For fixed tissues, perfusion with 4% paraformaldehyde followed by proper post-fixation is recommended.

• Protein extraction for Western blot:

  • Homogenize brain tissue in ice-cold lysis buffer containing protease inhibitors

  • For membrane protein enrichment, use a fractionation protocol to isolate membrane proteins

  • Include phosphatase inhibitors if studying phosphorylation states

  • Sonicate briefly and centrifuge at 14,000 × g for 15 minutes at 4°C

  • Collect supernatant and determine protein concentration

• Immunoprecipitation conditions: For co-immunoprecipitation studies investigating FXYD7 association with Na,K-ATPase, use mild non-denaturing conditions as described in the literature . This approach preserved the interaction between FXYD7 and Na,K-ATPase α subunits in research studies.

• Tissue section preparation for IHC/IF: For immunohistochemistry or immunofluorescence, 5-7 μm sections are typically used with standard antigen retrieval methods. Citrate buffer (pH 6.0) heat-induced epitope retrieval has shown good results for FXYD7 detection .

The critical consideration is preserving the native interactions of FXYD7 with Na,K-ATPase complexes while maintaining adequate protein extraction efficiency.

How should researchers address specificity concerns when working with FXYD7 antibodies?

Antibody specificity is crucial when working with FXYD7 due to the existence of multiple FXYD family members with structural similarities. To address specificity concerns:

• Validate with positive and negative controls:

  • Positive control: Brain tissue samples (especially cerebral cortex) where FXYD7 is highly expressed

  • Negative control: Kidney, liver, or other non-brain tissues where FXYD7 is not expressed

  • Consider using recombinant FXYD7 protein as a positive control

• Perform cross-reactivity testing:

  • Perform Western blots on tissues expressing other FXYD family members

  • Compare staining patterns with published FXYD7 localization data

  • Consider pre-absorption tests with immunizing peptide to confirm specificity

• Verify with orthogonal methods:

  • Complement antibody-based detection with mRNA analysis (RT-PCR, in situ hybridization)

  • Use multiple antibodies recognizing different epitopes of FXYD7

  • For critical findings, consider genetic approaches (knockdown/knockout) to confirm specificity

• Documentation and reporting:

  • Record antibody catalog numbers, lot numbers, and dilution factors

  • Document all validation steps performed and controls used

  • Report any cross-reactivity observed with other FXYD family members

Research has shown that commercial FXYD7 antibodies typically do not cross-react with other members of the FXYD protein family when properly validated , but verification in your specific experimental system is always recommended.

How can researchers optimize immunofluorescence protocols to detect both FXYD7 and Na,K-ATPase α subunits in brain sections?

Co-localization studies of FXYD7 with Na,K-ATPase α subunits require careful optimization of double immunofluorescence protocols. Based on published research methodologies , the following approach is recommended:

• Antibody selection and validation:

  • Choose FXYD7 and Na,K-ATPase α subunit antibodies raised in different host species

  • Verify each antibody individually before attempting co-localization

  • Ensure antibodies recognize the target proteins in their native conformation

• Optimized protocol for double immunofluorescence:

  • Fix brain tissue in 4% paraformaldehyde

  • Prepare 5-7 μm sections and perform antigen retrieval (citrate buffer pH 6.0)

  • Block with 5-10% normal serum from species not related to either primary antibody

  • Apply primary antibodies sequentially or simultaneously (after validation)

  • Use fluorophore-conjugated secondary antibodies with minimal spectral overlap

  • Include DAPI staining for nuclear visualization

  • Mount with anti-fade medium to preserve fluorescence

• Controls for co-localization studies:

  • Single-antibody controls to assess bleed-through

  • Secondary-only controls to assess non-specific binding

  • Peptide competition controls for specificity verification

• Advanced imaging considerations:

  • Use confocal microscopy for accurate co-localization assessment

  • Perform z-stack acquisition to ensure true co-localization in three dimensions

  • Consider spectral unmixing for closely overlapping fluorophores

  • Use appropriate co-localization analysis software and statistical methods

Research has demonstrated co-localization of FXYD7 with synaptophysin and GFAP, indicating its presence in both neurons and astroglial cells, with predominant expression in neurons . Similar approaches can be applied to studying FXYD7 co-localization with different Na,K-ATPase α subunit isoforms.

What strategies can resolve discrepancies in FXYD7-Na,K-ATPase interaction data between different experimental systems?

Researchers sometimes observe differences in FXYD7-Na,K-ATPase interactions between heterologous expression systems and native brain tissue. To address these discrepancies:

• Systematic comparison of experimental systems:

  • Compare results from native brain tissue, primary neuronal cultures, and heterologous expression systems

  • Document differences in expression levels, post-translational modifications, and interacting partners

  • Consider the influence of detergent types and concentrations on preserving protein-protein interactions

• Analysis of isoform-specific interactions:

  • In heterologous systems (e.g., Xenopus oocytes), FXYD7 can interact with Na,K-ATPase α1–β1, α2–β1, and α3–β1 isozymes but not with α–β2 isozymes

  • In brain tissue, FXYD7 was found to associate primarily with α1–β isozymes but not with α2–β or α3–β complexes

  • These differences may reflect cell-type specific regulation or experimental limitations

• Methodological reconciliation approaches:

  • Use crosslinking agents to stabilize transient interactions

  • Apply proximity ligation assays for in situ detection of protein interactions

  • Combine co-immunoprecipitation with mass spectrometry for unbiased identification of interacting partners

  • Consider native gel electrophoresis to preserve protein complexes

• Functional correlation analysis:

  • Correlate biochemical interaction data with functional measurements (e.g., Na,K-ATPase activity assays)

  • Assess how experimental conditions affect both protein interactions and functional outcomes

  • Develop mathematical models to reconcile divergent experimental findings

Studies have shown that while FXYD7 can interact with multiple Na,K-ATPase α–β1 isozymes in heterologous systems, it appears to function predominantly through α1–β1 complexes in brain tissue . These findings underscore the importance of validating heterologous expression system results in the native tissue context.

How can researchers accurately measure the effect of FXYD7 on Na,K-ATPase kinetics in neuronal preparations?

To accurately measure FXYD7's effects on Na,K-ATPase kinetics in neuronal preparations:

• Preparation of neuronal membrane fractions:

  • Isolate neuronal membranes through differential centrifugation

  • Prepare synaptosomes for studies focused on presynaptic Na,K-ATPase

  • Consider using gradient separation for purity enhancement

• Na,K-ATPase activity assays:

  • Ouabain-sensitive ATPase activity: Measure ATP hydrolysis in the presence and absence of ouabain

  • Rb+ uptake assays: Use 86Rb+ as a K+ congener to measure transport activity

  • Electrophysiological measurements: Patch-clamp recordings of pump currents

• Kinetic analysis approaches:

  • Measure Na+ and K+ concentration-dependent activation curves

  • Determine Km and Vmax values through Michaelis-Menten kinetics

  • Assess voltage dependence of Na,K-ATPase activity when FXYD7 is present

• Experimental manipulation of FXYD7:

  • Use FXYD7 knockdown/knockout approaches to assess loss-of-function effects

  • Apply FXYD7 overexpression systems to evaluate gain-of-function outcomes

  • Consider FXYD7 mutants lacking post-translational modifications

Published research has demonstrated that FXYD7 decreases the apparent K+ affinity of Na,K-ATPase α1–β1 and α2–β1 isozymes approximately 2-fold, without affecting α3–β1 isozymes . This selective modulation suggests FXYD7 adapts the transport properties of Na,K-ATPase to meet tissue-specific physiological requirements in the brain.

What is the current understanding of FXYD7's role in neuronal excitability and potential implications for neurological disorders?

The current understanding of FXYD7's role in neuronal excitability and potential implications for neurological disorders can be summarized as follows:

• Physiological role in neuronal excitability:

  • By decreasing the apparent K+ affinity of Na,K-ATPase α1–β1 isozymes, FXYD7 modifies the ion transport properties crucial for maintaining neuronal membrane potential

  • This modification may fine-tune neuronal excitability in a brain region-specific manner

  • FXYD7's brain-specific expression pattern suggests specialized roles in neuronal function not required in other tissues

• Cellular distribution and functional implications:

  • FXYD7 is present in both neurons and astrocytes, with predominant expression in neurons

  • The presence in astrocytes suggests potential roles in glial K+ buffering and neuron-glia interactions

  • Regional distribution within the brain (high in various regions, lowest in hypothalamus) may reflect region-specific requirements for Na,K-ATPase regulation

• Potential implications in neurological disorders:

  • Alterations in Na,K-ATPase function have been implicated in epilepsy, ischemia, and neurodegenerative disorders

  • As a regulator of Na,K-ATPase, FXYD7 may contribute to pathophysiological mechanisms in these conditions

  • Changes in FXYD7 expression or function could potentially affect neuronal excitability and contribute to seizure susceptibility or excitotoxicity

• Future research directions:

  • Investigate FXYD7 expression changes in animal models of neurological disorders

  • Assess the effects of FXYD7 genetic variants on Na,K-ATPase function and neuronal excitability

  • Explore potential therapeutic approaches targeting FXYD7-Na,K-ATPase interactions

The brain-specific expression of FXYD7 and its selective modulation of Na,K-ATPase isozymes suggest it plays an important role in adapting Na,K-ATPase function to the specialized requirements of neuronal signaling. Further research is needed to fully elucidate its role in normal brain function and potential contributions to neurological disorders.

How can advanced imaging techniques enhance our understanding of FXYD7 dynamics in living neurons?

Advanced imaging techniques offer powerful approaches to study FXYD7 dynamics in living neurons:

• Super-resolution microscopy applications:

  • STED (Stimulated Emission Depletion) microscopy: Enables visualization of FXYD7 distribution within membrane microdomains beyond the diffraction limit

  • STORM/PALM techniques: Allow single-molecule localization to map FXYD7 distribution with nanometer precision

  • Structured Illumination Microscopy (SIM): Provides enhanced resolution for studying FXYD7 co-localization with Na,K-ATPase subunits

• Live-cell imaging strategies:

  • Fluorescent protein tagging: Generate FXYD7-GFP fusion constructs for real-time visualization

  • FRAP (Fluorescence Recovery After Photobleaching): Assess FXYD7 lateral mobility within neuronal membranes

  • FLIM-FRET (Fluorescence Lifetime Imaging-Förster Resonance Energy Transfer): Measure dynamic interactions between FXYD7 and Na,K-ATPase subunits

• Correlative microscopy approaches:

  • Combine live-cell imaging with post-fixation immunolabeling

  • Implement CLEM (Correlative Light and Electron Microscopy) to correlate functional imaging with ultrastructural localization

  • Use neuronal activity indicators (e.g., calcium sensors) in parallel with FXYD7 imaging to correlate dynamics with neuronal activity

• Quantitative analysis methods:

  • Apply computational image analysis for quantitative assessment of FXYD7 distribution

  • Use single-particle tracking to study mobility of individual FXYD7 molecules

  • Develop mathematical models to relate FXYD7 dynamics to Na,K-ATPase function

These advanced imaging approaches can provide unprecedented insights into how FXYD7 dynamically regulates Na,K-ATPase in response to neuronal activity or pathological conditions, extending beyond the static images obtained with conventional immunofluorescence techniques that have demonstrated FXYD7's presence in neurons and astrocytes .

What considerations should researchers take into account when designing CRISPR/Cas9 approaches to study FXYD7 function?

When designing CRISPR/Cas9 approaches to study FXYD7 function, researchers should consider:

• Target design and validation considerations:

  • Guide RNA selection: Design multiple sgRNAs targeting exonic regions of FXYD7, avoiding regions with homology to other FXYD family members

  • Off-target prediction: Use computational tools to identify and avoid potential off-target sites

  • Validation strategy: Plan for sequencing-based validation of edits and assessment of potential off-target modifications

  • Functional domains: Consider targeting specific functional domains (e.g., the FXYD motif, transmembrane domain) for structure-function studies

• Delivery approaches for neuronal systems:

  • Primary neuronal cultures: Consider timing of transfection/transduction relative to neuronal maturation

  • In vivo approaches: Evaluate AAV-mediated delivery, in utero electroporation, or generation of transgenic models

  • Cell-type specificity: Use neuron-specific or astrocyte-specific promoters for targeted expression

• Experimental design for functional assessment:

  • Complete knockout vs. knockdown: Consider physiological consequences of complete FXYD7 elimination versus partial reduction

  • Temporal control: Implement inducible CRISPR systems for developmental stage-specific manipulation

  • Rescue experiments: Design complementation studies with wild-type or mutant FXYD7 to confirm specificity

  • Phenotypic analysis: Plan comprehensive assessment of Na,K-ATPase function, neuronal excitability, and behavior

• Special considerations for FXYD7:

  • Small gene size: The small size of FXYD7 (single exon coding region) may limit sgRNA design options

  • Post-translational modifications: Consider approaches to specifically target regions important for O-glycosylation

  • Potential compensation: Assess possible compensatory upregulation of other FXYD family members

  • Brain region specificity: Consider region-specific CRISPR delivery to match the differential expression of FXYD7 across brain regions

By carefully considering these factors, researchers can develop effective CRISPR/Cas9 approaches to study the function of FXYD7 in regulating Na,K-ATPase in the brain, potentially revealing new insights into its role in neuronal physiology and pathophysiology.