Phospho-ATP1A1 (Ser23) Antibody

What is Phospho-ATP1A1 (Ser23) Antibody and what epitope does it recognize?

Phospho-ATP1A1 (Ser23) Antibody is a primary polyclonal antibody specifically designed to detect the alpha-1 subunit of Na+/K+ ATPase (ATP1A1) only when phosphorylated at serine 23. The antibody recognizes the phosphorylated epitope around the Ser23 residue, typically within a peptide sequence of K-K-G(p)-K-K in the N-terminal domain of ATP1A1 . This specificity makes it valuable for studying post-translational regulation of Na+/K+ ATPase activity through PKC-mediated phosphorylation .

What are the validated applications for Phospho-ATP1A1 (Ser23) Antibody?

Based on current literature and commercial validation data, this antibody has been successfully employed in multiple experimental techniques:

• Western Blotting (WB): Validated at dilutions of 1:500-1:2000

• Immunofluorescence/Immunocytochemistry (IF/ICC): Effective at dilutions of 1:100-1:500

• Enzyme-Linked Immunosorbent Assay (ELISA): Works at dilutions of 1:2000-1:10000

• Cell-Based Phosphorylation ELISA: Specialized kits available for detecting phosphorylated ATP1A1 in intact cells

What species reactivity has been confirmed for this antibody?

The antibody demonstrates confirmed reactivity with:

• Rat: Most extensively validated across multiple suppliers

• Human: Some antibody preparations show cross-reactivity

• Mouse: Limited validation but reported by some manufacturers

When selecting this antibody for experiments with specific species, verification of cross-reactivity with your particular tissue or cell type is recommended, as reactivity can vary between manufacturers .

How should I design control experiments when using Phospho-ATP1A1 (Ser23) Antibody?

Robust experimental design with this antibody should include multiple controls:

• Peptide competition assay: Pre-incubate the antibody with the immunizing phosphopeptide to confirm signal specificity. Western blot images show complete signal abolishment when the antibody is blocked with the phosphopeptide .

• Non-phosphorylated control: Compare samples with and without treatments that induce Ser23 phosphorylation (e.g., PKC activators like phorbol esters) .

• Total ATP1A1 normalization: Use a separate antibody against total ATP1A1 (regardless of phosphorylation state) on parallel samples to normalize phosphorylation levels .

• Loading control: Include GAPDH detection as an internal control for normalization, especially in cell-based ELISA formats .

• Negative controls: Include secondary antibody-only controls to assess background signals .

What are the optimal sample preparation methods for detecting phosphorylated ATP1A1?

For successful detection of phosphorylated ATP1A1 at Ser23:

• Tissue/cell lysis: Use phosphatase inhibitor-containing buffers (e.g., phosphate buffered saline without Mg²⁺ and Ca²⁺, pH 7.4, with phosphatase inhibitor cocktail) to prevent dephosphorylation during sample preparation .

• Brain tissue processing: Several validations used rat brain tissue, suggesting this as an optimal source with abundant phosphorylated target .

• Cell fixation for immunofluorescence: For adherent cells, 4% formaldehyde is recommended; for suspension cells, 8% formaldehyde provides better results .

• Protein denaturation: Standard SDS-PAGE conditions with reducing agents are suitable for Western blot applications, targeting the expected molecular weight of approximately 113 kDa .

• Storage conditions: After sample collection, immediate processing or storage at -80°C is recommended to preserve phosphorylation states .

What treatments can modulate ATP1A1 Ser23 phosphorylation for experimental studies?

Several approaches can be used to experimentally modulate ATP1A1 phosphorylation at Ser23:

• PKC activators: Phorbol esters (like PDBu) have been shown to induce phosphorylation of Ser23, making them useful positive controls .

• Arachidonic acid: This can mimic phorbol ester effects on Na+/K+ ATPase activity by activating the PKC pathway .

• Cytochrome P450-dependent monooxygenase inhibitors: These can block the downstream effects of PKC activation on Na+/K+ ATPase .

• Temperature modulation: Incubation of cells at 18°C vs. 37°C affects the regulation patterns of Na+/K+ ATPase after PKC stimulation .

• Phospholipase A₂ inhibitors: These can influence the phosphorylation pathway that affects ATP1A1 .

How can I properly normalize and quantify Phospho-ATP1A1 (Ser23) signals?

For accurate quantification of phosphorylation signals:

• Ratio to total protein: Calculate the ratio of phospho-ATP1A1 to total ATP1A1 using the formula:
  OD₄₅₀ (Anti-ATP1A1 P-Ser23 Antibody)/OD₄₅₀ (Anti-ATP1A1 Antibody)

• GAPDH normalization: For cell-based assays, normalize phospho-signals to GAPDH as an internal loading control .

• Cell density normalization: In cell-based ELISAs, Crystal Violet staining can be used to normalize signals to cell number using the proportion OD₄₅₀/OD₅₉₅ .

• Multiple sample analysis: When comparing treatment effects, analyze samples in triplicate and perform statistical analysis to determine significance .

• Western blot quantification: Use densitometry software to quantify band intensities, normalizing phospho-signal to total protein or housekeeping controls .

What is the relationship between ATP1A1 Ser23 phosphorylation and enzyme activity?

Understanding this relationship is critical for functional studies:

• Activity inhibition: Phosphorylation of ATP1A1 at Ser23 by protein kinase C has been demonstrated to decrease the activity of Na+/K+ ATPase in vitro .

• Conformational shift mechanism: Ser23 phosphorylation results in a shift in equilibrium toward the E1 form of the enzyme, as measured by eosin fluorescence studies .

• Potassium affinity: This phosphorylation is associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays .

• Tissue-specific effects: The inhibitory effect observed in thick ascending limb cells contrasts with effects in proximal tubules, suggesting context-dependent regulation .

• Regulatory domain: The NH2-terminal domain of the alpha-1 subunit, including Ser23, plays a critical role in regulating enzyme activity, as supported by tryptic digestion studies .

How does ATP1A1 Ser23 phosphorylation differ from other phosphorylation sites on the protein?

ATP1A1 contains multiple phosphorylation sites with distinct functions:

• Ser23 vs. Ser16: While Ser23 phosphorylation generally inhibits Na+/K+ ATPase activity, phosphorylation at Ser16 has been shown to have a stimulatory effect, increasing the apparent Na+ affinity of the enzyme .

• Multiple regulatory sites: The alpha-1 subunit can be phosphorylated at multiple residues including Ser11, Ser18, Ser23, and Ser938, each potentially having different regulatory effects .

• PKC vs. cAMP-dependent phosphorylation: PKC primarily targets Ser23, while cAMP-dependent phosphorylation targets other residues and increases activity in thick ascending limb cells .

• Tissue-specific effects: The same phosphorylation site can have opposite effects in different cell types; Ser23 phosphorylation inhibits activity in thick ascending limbs but has different effects in proximal tubules .

• Signaling pathway integration: The net effect on Na+/K+ ATPase activity depends on the integration of multiple phosphorylation events and their tissue-specific contexts .

What strategies can overcome detection challenges with Phospho-ATP1A1 (Ser23) Antibody?

When facing difficulties in phosphorylation detection:

• Signal enhancement methods:

  • For Western blots: Use high-sensitivity ECL substrates and longer exposure times

  • For immunofluorescence: Consider tyramide signal amplification (TSA) techniques

  • For ELISA: Extended substrate incubation times may improve detection

• Phosphatase inhibitor optimization: Include multiple phosphatase inhibitors (serine/threonine and tyrosine) in extraction buffers to prevent dephosphorylation during sample preparation .

• Antibody purification method verification: Confirm the antibody was purified by sequential chromatography on phospho-peptide and non-phospho-peptide affinity columns to ensure phospho-specificity .

• Antigen retrieval techniques: For fixed tissue samples, optimize antigen retrieval methods to improve epitope accessibility .

• Cell type considerations: Based on validation data, neuronal tissues like rat brain show strong phospho-ATP1A1 signals and may serve as positive controls .

How can I establish functional significance of ATP1A1 Ser23 phosphorylation in my experimental system?

To connect phosphorylation with functional outcomes:

• Activity assays: Combine phosphorylation detection with Na+/K+ ATPase activity assays to correlate modification levels with enzymatic function .

• Mutagenesis approach: Utilize site-directed mutagenesis to create S23A (phospho-null) and S23D/E (phospho-mimetic) variants for functional studies in transfected cells .

• Eosin fluorescence studies: This technique can monitor conformational changes between E1 and E2 states to understand how phosphorylation affects enzyme conformation .

• K+ affinity measurement: Design experiments to measure K+ dependency of ATPase activity in phosphorylated versus non-phosphorylated samples .

• Physiological outcome assessment: In specialized cells like those in the thick ascending limb, measure downstream effects like ion transport to correlate with phosphorylation status .

What are the critical methodological differences between cell-based and standard ELISA approaches for Phospho-ATP1A1 (Ser23) detection?

Understanding these methodological distinctions is essential for choosing the appropriate technique:

• Sample preparation differences:

  • Cell-based: Cells are fixed directly in the plate with formaldehyde (4% for adherent, 8% for suspension cells)

  • Standard ELISA: Requires cell lysis, protein extraction, and plate coating

• Sensitivity comparison:

  • Cell-based: Can detect phosphorylated ATP1A1 in as few as 5,000 cells

  • Standard ELISA: Typically requires higher protein amounts but offers more precise quantification

• Normalization approaches:

  • Cell-based: Offers multiple normalization options (GAPDH, total ATP1A1, cell density via Crystal Violet)

  • Standard ELISA: Typically normalized to total protein concentration

• Workflow complexity:

  • Cell-based: 16-step protocol including fixing, blocking, primary and secondary antibody incubations

  • Standard ELISA: Requires additional initial steps for sample preparation and coating

• Data presentation:

  • Cell-based: Results typically presented as relative phosphorylation levels

  • Standard ELISA: Can provide more precise quantification with standard curves

How is Phospho-ATP1A1 (Ser23) involved in pathophysiological conditions?

Current research suggests several disease-relevant connections:

• Thick ascending limb transport disorders: Phosphorylation of ATP1A1 at Ser23 in thick ascending limb cells has implications for renal sodium handling and potentially hypertension pathogenesis .

• Na+/K+ ATPase regulation: As the phosphorylation inhibits pump activity, it may contribute to ionic imbalances in various pathological states requiring altered Na+/K+ transport .

• PKC signaling dysregulation: Conditions with altered PKC activity may show corresponding changes in ATP1A1 Ser23 phosphorylation, affecting cellular ion homeostasis .

• Neurological implications: Given the validation in brain tissues and the critical role of Na+/K+ ATPase in neuronal function, Ser23 phosphorylation may have relevance to neurological disorders .

• Integrated signaling networks: The phosphorylation status at Ser23 represents one node in complex signaling networks that regulate ion transport, with potential involvement in various disease states .

What advanced experimental systems can be used to study the physiological impact of ATP1A1 Ser23 phosphorylation?

Sophisticated experimental approaches include:

• Transgenic expression systems: Studies using COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo α1 subunits provide insights into phosphorylation effects .

• Temperature-controlled experiments: Incubating cells at different temperatures (18°C vs. 37°C) can reveal distinct regulatory mechanisms by suppressing trafficking effects at lower temperatures .

• Isolated thick ascending limb preparation: This specialized technique allows for the study of Ser23 phosphorylation effects in a physiologically relevant tubular segment .

• Electrophysiological measurements: Combining phosphorylation detection with patch-clamp or other electrophysiological techniques can directly link phosphorylation to functional outcomes.

• In vivo phosphorylation monitoring: Development of phospho-specific probes or biosensors could enable real-time monitoring of ATP1A1 phosphorylation states in living cells or tissues.

What technological developments are enhancing phosphorylation site-specific antibody applications?

Emerging technologies improving phospho-specific detection include:

• Multiplex phosphorylation detection: Advanced methods allow simultaneous detection of multiple phosphorylation sites (e.g., Ser16, Ser23, Tyr10) to understand integrated regulation .

• Super-resolution microscopy: These techniques provide subcellular localization information for phosphorylated ATP1A1, potentially revealing compartmentalized regulation.

• Phospho-proteomics integration: Combining antibody-based detection with mass spectrometry-based phospho-proteomics provides comprehensive phosphorylation profiles and stoichiometry information.

• Single-cell phosphorylation analysis: Adaptation of phospho-specific antibodies for single-cell analysis techniques reveals cell-to-cell variability in ATP1A1 regulation.

• Proximity labeling approaches: Techniques like BioID or APEX2 combined with phospho-specific antibodies can identify proteins interacting specifically with the phosphorylated form of ATP1A1.