FXYD1 Antibody

What tissue samples are most reliable for FXYD1 antibody validation?

According to validation data from multiple sources, FXYD1 antibodies show consistent detection in specific tissues. Commercially available antibodies have been tested across various sample types with positive results in:

For optimal antibody validation, human skeletal muscle and heart tissues represent the most consistent positive controls across multiple antibody products . These tissues demonstrate high endogenous expression levels, making them ideal for initial antibody characterization.

What are the recommended antibody dilutions for common applications?

Proper antibody dilution is essential for balancing specific signal against background. Based on validated protocols, the following dilution ranges are recommended for FXYD1 antibodies:

It is strongly recommended that each antibody be titrated in your specific testing system to obtain optimal results, as the ideal dilution may be sample-dependent . Sensitivity can vary significantly between tissues and experimental conditions.

How can I distinguish between phosphorylated and non-phosphorylated forms of FXYD1?

This represents a critical technical challenge when studying FXYD1 function. The phosphorylation status of FXYD1 significantly affects its biological activity and Na+/K+-ATPase regulation.

When using the AB_FXYD1 antibody, it's important to note that it predominantly recognizes unphosphorylated FXYD1. Phosphorylation at Ser-63, Ser-68, and Thr-69 reduces AB_FXYD1 signal intensity because the antibody epitope is located in the COOH-terminal region where the phosphorylation sites are located .

For detecting phosphorylated forms:

• Use phospho-specific antibodies targeting specific sites (e.g., pSer68)

• Employ dephosphorylation protocols to confirm specificity

• Validate with complementary approaches such as:

To confirm AB_FXYD1 phospho-specificity, researchers have developed a dephosphorylation validation protocol:

• Strip the membrane after initial Western blotting

• Confirm complete stripping by ECL exposure

• Incubate the membrane with dephosphorylation buffer (50 mM Tris·HCl, 0.1 mM Na2EDTA, 5 mM DTT, 0.01% Brij 35, 2 mM MnCl2; pH 7.5) containing lambda protein phosphatase (500 U/ml)

• Reblot with AB_FXYD1 antibody

This approach reveals the total FXYD1 expression and can demonstrate significant differences between phosphorylated and total FXYD1 levels.

What are the proven methods for studying FXYD1 glutathionylation?

FXYD1 glutathionylation plays an important role in regulating Na+/K+-ATPase function under oxidative stress conditions. Research has demonstrated that:

• Glutathionylation of FXYD1 can be detected at baseline in cardiomyocytes

• Exposure to angiotensin II (15 min) increases glutathionylation by activating cardiac NADPH oxidase

• The adenyl cyclase activator forskolin also increases glutathionylation of FXYD1

• Hypoxic conditions promote protein glutathionylation and inhibit glutaredoxin 1 (Grx1)

Methodologically, researchers have successfully assessed FXYD1 glutathionylation using:

• GSH antibody technique to detect glutathionylation at the time of cell lysis

• Biotin-GSH technique to estimate the proportion of glutathionylated protein

• Co-immunoprecipitation approaches followed by Western blotting with GSH antibodies

To confirm specificity, controls involving incubation with 1 μM recombinant human Grx1 or 1 mM DTT should be included, as these treatments eliminate the glutathionylation signal .

How can I evaluate FXYD1 and Na+/K+-ATPase interactions experimentally?

FXYD1 associates with and regulates the Na+/K+-ATPase, making this interaction crucial for understanding its physiological role. There are several validated approaches:

• Co-immunoprecipitation studies:

  • Immunoprecipitate with Na+/K+-ATPase α-subunit antibody and detect FXYD1 in the precipitate

  • Alternatively, immunoprecipitate with FXYD1 antibody and detect Na+/K+-ATPase subunits

  • For example, one study demonstrated that "FXYD1 was detected readily in total cell lysate with an FXYD1 antibody... It was also detected in α1 subunit immunoprecipitate"

• Expression systems:

  • Co-expression of FXYD1 with Na+/K+-ATPase subunits in Xenopus oocytes followed by functional studies

  • Research has demonstrated that "The expressed FXYD1 associates with the Na+-K+ pump as indicated by co-immunoprecipitation experiments"

  • This approach allows for mutagenesis studies to determine critical interaction domains

• Surface biotinylation techniques:

  • Surface-expressed FXYD proteins can be labeled using p-diazobenzoyl biocytin (DBB)

  • This method allows differentiation between total and surface-expressed protein

  • The protocol involves incubating cells with DBB, quenching with BSA, and isolating biotinylated proteins on streptavidin beads

What strategies are effective for studying FXYD1 trafficking in polarized cells?

FXYD1 trafficking is complex and cell-type dependent. Research shows that FXYD1 can be expressed in the plasma membrane only when coexpressed with Na+/K+-ATPase, while other FXYD family members like FXYD7 may reach the membrane independently .

For studying apical versus basolateral localization in polarized epithelial cells:

• Grow cells on Transwell inserts (pore size 0.4 μm) until confluent monolayers with transepithelial electric resistance >1 kΩ × cm² are established

• For selective surface biotinylation, add DBB (0.5 mg/ml) to either the basolateral (lower) or apical (upper) compartment

• Incubate for 30 minutes at 4°C, followed by washing with quenching buffer

• Process cells and analyze by Western blotting to determine domain-specific expression

For fluorescence-based trafficking studies:

• Create fluorescently tagged constructs (e.g., CFP-tagged FXYD1)

• Express in appropriate cell lines using transfection reagents like ICAFectin®441 or JetPei

• Visualize using confocal microscopy through an oil immersion objective at 37°C with CO₂ supply

Why might I observe variations in FXYD1 molecular weight across different samples?

FXYD1 has a calculated molecular weight of approximately 10 kDa (92 amino acids), but researchers frequently observe variations in the apparent molecular weight:

• The observed molecular weight typically ranges from 10-15 kDa

• Post-translational modifications affect migration patterns:

  • Phosphorylation at multiple sites (Ser-63, Ser-68, Thr-69)

  • Glutathionylation

  • Potential O-glycosylation (similar to FXYD7)

When comparing FXYD1 between different tissues or experimental conditions, these variations should be considered and potentially exploited to gain insights into post-translational modification states.

How can I address challenges when studying FXYD1 in knockout/knockdown models?

FXYD1⁻/⁻ mice have been valuable for studying FXYD1 function. Key findings include:

• Increased β1 subunit glutathionylation in FXYD1⁻/⁻ myocardium compared to wild-type

• Altered Na+/K+-ATPase regulation under oxidative stress conditions

When designing experiments with these models:

• Always validate antibody specificity using knockout tissues

• Include appropriate wild-type littermate controls

• Consider compensatory changes in other FXYD family members

• Address tissue-specific effects, as FXYD1 function may vary between cardiac, skeletal muscle, and other tissues

For knockdown approaches, researchers should verify efficiency at both protein and mRNA levels, as post-transcriptional regulation may affect FXYD1 protein abundance independently of mRNA levels.

What antigen retrieval methods are most effective for FXYD1 immunohistochemistry?

Antigen retrieval is critical for successful FXYD1 immunohistochemistry. Based on validated protocols:

• TE buffer pH 9.0 is recommended as the primary antigen retrieval method

• Alternatively, citrate buffer pH 6.0 may be used, though possibly with reduced sensitivity

For optimal results with paraffin-embedded human cardiac muscle tissue:

• Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0

• Use recommended antibody dilutions (typically 1:50-1:500)

• Follow with appropriate detection systems (e.g., pre-diluted HRP Polymer for Rabbit IgG secondary antibody)

• Include appropriate negative controls (PBS substitution for primary antibody)

This approach has been validated to produce specific membrane staining patterns in cardiac tissue samples.

How might newer FXYD1 antibody technologies enhance understanding of cardiac and skeletal muscle physiology?

Emerging research suggests several promising directions:

• Development of conformation-specific antibodies that distinguish between FXYD1 bound to Na+/K+-ATPase versus free FXYD1

• Creation of FXYD1 antibodies that recognize specific phosphorylation patterns across all three major sites (Ser-63, Ser-68, Thr-69)

• Application of nanobody technology for real-time imaging of FXYD1 trafficking and interactions

These approaches could significantly enhance our understanding of FXYD1's dynamic regulation in response to hormonal stimulation, oxidative stress, and exercise.

What are the potential applications of FXYD1 antibodies in studying disease mechanisms?

FXYD1 has been implicated in several pathological conditions, particularly those involving altered ion homeostasis. Future research applications include:

• Investigating FXYD1 expression and phosphorylation status in heart failure models

• Examining the relationship between FXYD1 glutathionylation and cardiac ischemia-reperfusion injury

• Exploring FXYD1's role in skeletal muscle adaptations to exercise and disease

• Studying FXYD1 contributions to neuronal excitability disorders

Antibodies that can distinguish between different post-translational modification states will be particularly valuable for these investigations.