ATP8A1 Antibody

What is ATP8A1 and what is its primary function in cells?

ATP8A1 is a member of the P4-ATPase family of phospholipid flippases that specifically translocates phosphatidylserine (PS) from the outer/luminal leaflet to the inner/cytosolic leaflet of cellular membranes . This protein forms a functional complex with CDC50A, which serves as its essential β-subunit . ATP8A1 plays a critical role in maintaining phospholipid asymmetry, which is fundamental for multiple cellular processes including:

• Cell migration and motility

• Regulation of endosomal trafficking pathways

• Modulation of receptor signaling, particularly EGFR degradation and signaling

ATP8A1 functions primarily at the endosomal level rather than at the plasma membrane, with particular enrichment in Rab7-positive late endosomal compartments .

What is the molecular structure and biochemical properties of ATP8A1?

ATP8A1 has the following key structural and biochemical characteristics:

• Full-length protein consists of 1164 amino acids

• Calculated molecular weight of approximately 131 kDa

• Contains ATPase domains characteristic of P-type ATPases

• Forms a functional heterodimeric complex with CDC50A

• Requires ATP for its flippase activity

• Gene ID (NCBI): 10396

• UniProt accession: Q9Y2Q0

The protein likely has regulatory N- and C-terminal extensions, similar to other P4-ATPases like ATP8B1, with the C-terminus extending through its cytosolic catalytic domains .

What are the key applications for ATP8A1 antibodies in research?

ATP8A1 antibodies serve multiple research applications, including:

• Protein detection and quantification:

  • Western blotting for expression level analysis

  • Flow cytometry for quantitative assessment of surface vs. total expression

• Subcellular localization studies:

  • Immunofluorescence microscopy to determine intracellular distribution

  • Co-localization studies with endosomal markers (e.g., Rab5, Rab7, Rab11)

• Functional studies:

  • Validation of knockdown/knockout efficiency in siRNA or shRNA experiments

  • Confirmation of overexpression in gain-of-function studies

• Multi-parameter analysis:

  • Cytometric bead arrays for quantitative protein analysis

  • ELISAs for detection in complex biological samples

• Tissue expression profiling:

  • Immunohistochemistry for distribution in normal and diseased tissues

When selecting an antibody, researchers should consider validated applications for their specific experimental needs. For example, matched antibody pairs (like 60828-3-PBS capture and 60828-4-PBS detection) are specifically validated for cytometric bead array applications .

How should I validate the specificity of an ATP8A1 antibody?

Rigorous validation is essential for ensuring antibody specificity. Recommended validation approaches include:

• Genetic approaches:

  • Use siRNA or shRNA knockdown of ATP8A1 and confirm reduced signal by immunoblotting or immunofluorescence

  • Include ATP8A1 knockout cells/tissues as negative controls

• Expression system controls:

  • Compare signals between cells with endogenous vs. overexpressed ATP8A1

  • Test antibody against recombinant ATP8A1 protein

• Cross-reactivity assessment:

  • Test reactivity against related P4-ATPases (ATP8A2, ATP8B1, ATP11A)

  • Perform protein array analysis (as done with Prestige Antibodies against 364 human recombinant protein fragments)

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Compare blocked vs. unblocked antibody signals

• Multiple detection methods:

  • Confirm findings using different techniques (e.g., western blot, immunofluorescence, flow cytometry)

  • Ideally, use multiple antibodies targeting different epitopes

Researchers should document that antibody specificity was confirmed by "immunoblotting and immunofluorescence staining where the expression of endogenous proteins was suppressed by introducing siRNA," as described for custom antibodies .

What experimental approaches are recommended for studying ATP8A1's enzymatic activity?

ATP8A1's flippase activity can be assessed through several complementary approaches:

• ATPase activity assays:

  • Measure ATP hydrolysis using purified protein reconstituted in proteoliposomes

  • Quantify inorganic phosphate release using colorimetric methods (e.g., Malachite Green assay)

  • Include appropriate controls: vector-only preparations, catalytic mutants, and specific inhibitors

  • Example protocol: Incubate vesicles (2 μg protein) in buffer containing 50 mM HEPES-NaOH, 150 mM NaCl, 12.5 mM MgCl₂, 1 mM EGTA, and 10 mM ATP; measure phosphate release using Malachite Green dye and spectrophotometric detection at 650 nm

• Fluorescent phospholipid translocation assays:

  • Use fluorescently labeled PS (e.g., NBD-PS) in reconstituted systems

  • Track fluorescence changes upon translocation between membrane leaflets

  • Analyze by fluorescence spectroscopy or microscopy

• Cellular PS distribution analysis:

  • Use annexin V binding to detect PS exposure on cell surfaces

  • Compare wild-type cells with ATP8A1-depleted or overexpressing cells

  • Analyze by flow cytometry or microscopy

• Genetic complementation studies:

  • Test whether wild-type ATP8A1 can rescue phenotypes in ATP8A1-deficient cells

  • Include catalytically inactive mutants as negative controls

When interpreting results, consider that ATP8A1 primarily flips endosomal PS rather than plasma membrane PS , so experimental design should account for this compartment-specific activity.

How can I design experiments to investigate ATP8A1-CDC50A complex formation?

The ATP8A1-CDC50A interaction is crucial for proper flippase function. To study this complex:

• Co-immunoprecipitation approaches:

  • Use antibodies against ATP8A1 to pull down CDC50A or vice versa

  • Western blot to detect both proteins in the immunoprecipitate

  • Include appropriate controls (e.g., IgG control, single-transfected cells)

• Genetic manipulation strategies:

  • Generate knockdown/knockout of CDC50A to study effects on ATP8A1 stability, localization, and function

  • Create ATP8A1 mutants in potential CDC50A interaction sites

  • Test complementation with wild-type and mutant constructs

• Co-localization studies:

  • Perform dual-color immunofluorescence for ATP8A1 and CDC50A

  • Analyze by confocal microscopy and quantify co-localization coefficients

  • Compare wild-type and mutant proteins

• Tagged protein approaches:

  • Express differentially tagged versions (e.g., GFP-ATP8A1 and FLAG-CDC50A)

  • Perform pull-down experiments using anti-tag antibodies

  • Visualize interaction by proximity ligation assay

• Biochemical purification:

  • Co-express ATP8A1 and CDC50A and purify the complex

  • Analyze by size exclusion chromatography to confirm complex formation

  • Perform mass spectrometry to identify additional interacting partners

Reference describes useful methodology, including the creation of stable cell lines expressing GFP and HA-tagged ATP8A1, FLAG-tagged CDC50A, which can be adapted for complex formation studies.

How does ATP8A1 depletion affect endosomal sorting and signaling pathways?

ATP8A1 depletion has significant effects on endosomal trafficking and signaling, which can be investigated through:

• Receptor trafficking analysis:

  • Track EGFR degradation kinetics following EGF stimulation in ATP8A1-depleted cells

  • Research shows ATP8A1 knockdown enhances EGFR degradation by approximately 1.96-fold at 15 min, 1.18-fold at 30 min, and 0.67-fold at 60 min post-EGF treatment

  • Use cycloheximide to block protein synthesis during experiments

  • Verify lysosomal involvement using Bafilomycin A1 (BafA1) treatment

• Signaling pathway alterations:

  • Monitor phosphorylation status of downstream effectors (AKT, ERK)

  • ATP8A1 knockdown increases AKT activation at early time points but accelerates deactivation to basal levels; ERK phosphorylation is only marginally affected

  • Use phospho-specific antibodies and western blotting with careful time-course analysis

• Intraluminal vesicle (ILV) formation studies:

  • ATP8A1 depletion accelerates cargo protein transfer into ILVs of multivesicular bodies (MVBs)

  • Use electron microscopy to visualize ILV formation

  • Track cargo protein sorting into ILVs using fluorescently tagged proteins

• Phosphatidylserine distribution analysis:

  • ATP8A1 depletion leads to PS loading in the luminal leaflet of MVB limiting membranes

  • Use PS-specific probes to visualize PS distribution in endosomal compartments

  • Compare control and ATP8A1-depleted cells

• ESCRT recruitment studies:

  • Analyze recruitment of ESCRT components to endosomes in ATP8A1-depleted cells

  • Use immunofluorescence or live-cell imaging with tagged ESCRT proteins

  • Quantify ESCRT recruitment kinetics and levels

When designing these experiments, it's important to include both loss-of-function (shRNA/siRNA) and gain-of-function (overexpression) approaches, as these have opposite effects on EGFR degradation kinetics .

What controls should be included when studying ATP8A1's role in cell migration?

When investigating ATP8A1's role in cell migration, include these critical controls:

• Knockdown/knockout validation:

  • Confirm ATP8A1 depletion by western blot and qRT-PCR

  • Use multiple siRNA/shRNA sequences to rule out off-target effects

  • Include scrambled/non-targeting siRNA controls

• Rescue experiments:

  • Re-express siRNA-resistant ATP8A1 constructs in depleted cells

  • Include catalytically inactive mutants to demonstrate enzyme activity dependence

  • Test CDC50A co-expression requirement for function

• Migration assay controls:

  • Include positive controls (e.g., cells treated with migration-promoting factors)

  • Include negative controls (e.g., cytochalasin D to block actin polymerization)

  • Perform parallel proliferation assays to distinguish migration from proliferation effects

• Mechanistic controls:

  • Monitor key regulators of cell migration (e.g., Rac1 activation)

  • Track phosphatidylserine distribution using annexin V or PS-specific probes

  • Examine cytoskeletal reorganization during migration

• Alternative migration assays:

  • Use multiple migration assay formats (e.g., wound healing, transwell, single-cell tracking)

  • Compare 2D vs. 3D migration models

  • Test both random and directed migration

Reference indicates that ATP8A1 forms a complex with CDC50A that plays a major role in cell migration, making CDC50A status an essential control in these experiments.

How can apparent contradictions in ATP8A1 localization data be reconciled?

Different studies report varying subcellular localizations for ATP8A1. To address these contradictions:

• Comprehensive compartment analysis:

  • Perform systematic co-localization with markers for multiple compartments:

    • Early endosomes (Rab5)

    • Recycling endosomes (Rab11)

    • Late endosomes (Rab7)

    • Lysosomes (Lysotracker)

    • Golgi (GM130)

    • ER (calnexin)

    • Plasma membrane (WGA)

  • Quantify co-localization with each marker

  Compartment	Marker	Co-localization with ATP8A1
  Late endosomes	Rab7	Highest enrichment
  Early endosomes	Rab5	Moderate
  Recycling endosomes	Rab11	Moderate
  Lysosomes	Lysotracker	Moderate
  Plasma membrane	WGA	Very low
  Golgi	GM130	Low
  ER	Calnexin	Negligible

• Expression level considerations:

  • Compare endogenous vs. overexpressed protein localization

  • Ensure tagged constructs are expressed at low levels to avoid mislocalization

  • Use inducible expression systems to control expression levels

• Tissue-specific variation analysis:

  • Compare ATP8A1 localization across different cell types and tissues

  • ATP8A1 shows different expression patterns across tissues (kidney, testes, brain)

  • In retina, ATP8A1 shows distinct distribution compared to ATP8A2

• Dynamic trafficking studies:

  • Track ATP8A1 localization over time using live-cell imaging

  • Examine responses to stimuli that might alter trafficking

  • Consider that steady-state localization may not reflect dynamic behavior

• Detection method comparisons:

  • Compare results from different techniques (e.g., biochemical fractionation vs. microscopy)

  • Use multiple antibodies targeting different epitopes

  • Apply super-resolution microscopy for more detailed localization

The apparent contradictions may reflect cell type-specific differences, dynamic trafficking between compartments, or technical variations in detection methods.

How might post-translational modifications regulate ATP8A1 activity?

Post-translational modifications (PTMs) likely play important roles in regulating ATP8A1, based on studies of related P4-ATPases:

• Phosphorylation:

  • Investigate potential phosphorylation sites using phospho-specific antibodies

  • Studies of the related ATP8B1 show that phosphorylation at S1223 affects autoinhibition by the C-terminal tail

  • Design experiments to identify kinases and phosphatases that regulate ATP8A1

  • Use phosphomimetic (S→D/E) and phosphodeficient (S→A) mutations to study functional effects

• Autoinhibitory mechanisms:

  • The C-terminus of ATP8A1 extends through cytosolic catalytic domains, similar to ATP8A2

  • Design truncation mutants to test potential autoinhibitory roles of N- and C-termini

  • Create chimeric constructs swapping regulatory domains with other P4-ATPases

• Lipid interactions:

  • Investigate if phosphoinositides regulate ATP8A1 activity, as they do for ATP8B1

  • Design binding assays to identify regulatory lipid interactions

  • Use lipidomic approaches to characterize lipid environments of ATP8A1

• Ubiquitination:

  • Examine ubiquitination status of ATP8A1 in different conditions

  • Investigate if ubiquitination affects ATP8A1 stability, localization, or activity

  • Test effects of proteasome inhibitors on ATP8A1 levels and function

• Experimental approaches:

  • Immunoprecipitation followed by mass spectrometry to identify PTMs

  • Site-directed mutagenesis of potential modification sites

  • In vitro enzymatic assays with modified and unmodified proteins

  • Pharmacological inhibitors of modification enzymes

For related P4-ATPases, a cooperative mechanism involving N- and C-terminal extensions regulates activity, with phosphorylation altering this regulation . Similar mechanisms may apply to ATP8A1.

What are the optimal conditions for using ATP8A1 antibodies in immunofluorescence studies?

For successful immunofluorescence studies of ATP8A1:

• Fixation and permeabilization:

  • Test multiple fixation methods (4% paraformaldehyde, methanol, or combined approaches)

  • Optimize permeabilization conditions (0.1-0.5% Triton X-100, saponin, or digitonin)

  • Gentle permeabilization may better preserve membrane structures where ATP8A1 resides

• Antibody selection and validation:

  • Confirm antibody specificity through ATP8A1 knockdown controls

  • Consider using tagged ATP8A1 (GFP-ATP8A1, HA-ATP8A1) for detection with anti-tag antibodies

  • When available, use antibodies raised against different epitopes to confirm localization patterns

• Co-localization studies:

  • Include markers for endosomal compartments (Rab5, Rab7, Rab11)

  • Use high-resolution confocal or super-resolution microscopy

  • Perform quantitative co-localization analysis (Pearson's coefficient, Manders' overlap)

• Controls and quantification:

  • Include ATP8A1-depleted cells as negative controls

  • Quantify localization across multiple cells and experiments

  • Analyze distribution patterns using line scan profiles across cellular compartments

• Special considerations:

  • Flow cytometry analysis can complement immunofluorescence to quantify surface vs. total expression

  • For live-cell imaging, consider photobleaching approaches to study dynamics

  • When examining ATP8A1-CDC50A interactions, co-stain for both proteins

Reference provides a methodological framework, showing that permeabilized samples reveal total ATP8A1 expression while surface staining shows minimal plasma membrane localization.

What are the best experimental designs for studying ATP8A1 in knockout/knockdown models?

When designing ATP8A1 knockout/knockdown experiments:

• RNA interference approaches:

  • Use multiple siRNA sequences targeting different regions of ATP8A1 mRNA

    • Example target sequences: nucleotides 1947-1971 (siRNA-1) and 2059-2083 (siRNA-2) of hamster ATP8A1

  • Include non-targeting siRNA controls

  • Establish stable knockdown cell lines using shRNA for long-term studies

    • Example target sequence: nucleotides 2673-2695 of hamster ATP8A1

• CRISPR/Cas9 knockout strategies:

  • Design guide RNAs targeting early exons or essential domains

  • Screen multiple clones to identify complete knockouts

  • Sequence genomic DNA to confirm mutations

  • Create conditional knockout models for essential genes

• Validation approaches:

  • Confirm knockdown/knockout by western blot, qRT-PCR

  • Perform functional assays (e.g., PS flipping activity)

  • Include rescue experiments with wild-type ATP8A1

• Phenotypic analyses:

  • Examine effects on:

    • Phospholipid asymmetry using annexin V binding

    • Receptor trafficking and degradation (e.g., EGFR)

    • Endosomal morphology and dynamics

    • Cell migration

    • Signaling pathway activation (AKT, ERK)

• Important controls:

  • Include parallel experiments with other P4-ATPase family members (ATP8A2, ATP11A)

  • Consider potential compensatory upregulation of related flippases

  • For tissue-specific studies, consider the expression patterns of ATP8A1 across tissues

For comprehensive phenotypic analysis, reference demonstrates how ATP8A1 depletion affects EGFR trafficking and signaling, providing a methodological framework that can be adapted to other receptor systems.

How can I determine if an ATP8A1 antibody cross-reacts with other P4-ATPase family members?

Ensuring antibody specificity within the P4-ATPase family requires:

• Sequence analysis and epitope mapping:

  • Align the immunogen sequence with other P4-ATPases to identify potential cross-reactivity

  • Focus on antibodies raised against unique regions of ATP8A1

  • For commercial antibodies, request epitope information from manufacturers

• Expression system testing:

  • Test antibody against cells expressing individual P4-ATPases (ATP8A1, ATP8A2, ATP8B1, ATP11A)

  • Use overexpression systems with tagged constructs as positive controls

  • Include knockout/knockdown controls for each P4-ATPase

• Immunoprecipitation followed by mass spectrometry:

  • Perform IP with the ATP8A1 antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Identify any co-precipitated P4-ATPases

• Comparative tissue analysis:

  • Test antibody in tissues with differential expression of P4-ATPases

  • For example, ATP8A1 and ATP8A2 show distinct distribution patterns in retina :

    • ATP8A1: inner segment, outer and inner plexiform layers, inner nuclear layer, ganglion cells

    • ATP8A2: outer segments, ganglion cell layer, weaker in inner segment layer, plexiform layers

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide and peptides from related P4-ATPases

  • Compare blocking efficiency to assess cross-reactivity

Given the structural similarity between P4-ATPases, careful validation is essential, especially when studying tissues where multiple family members are expressed.

What methodology is recommended for quantifying ATP8A1 expression levels across different tissues?

For accurate quantification of ATP8A1 across tissues:

• RNA-based approaches:

  • qRT-PCR using validated primer sets specific to ATP8A1

  • RNA-seq for genome-wide expression profiling

  • Include reference genes appropriate for cross-tissue comparisons

  • Normalize to tissue-specific housekeeping genes

• Protein-based approaches:

  • Western blotting with proper loading controls

  • Use recombinant ATP8A1 standards for absolute quantification

  • Consider ELISA or cytometric bead arrays for higher throughput

  • Mass spectrometry-based targeted proteomics

• Tissue preparation considerations:

  • Standardize tissue collection and processing protocols

  • Consider regional variation within organs

  • For retina and brain, microdissection may be necessary for regional analysis

• Visualization approaches:

  • Immunohistochemistry on tissue microarrays for comparative analysis

  • Quantitative immunofluorescence with internal standards

  • Whole-slide scanning and digital image analysis

• Data interpretation:

  • Consider relative abundance of ATP8A1 across tissues:

    • Higher in kidney, brain

    • Detectable in testes

  • Compare with other P4-ATPases (ATP8A2, ATP11A, ATP8B1) in the same tissues

  • Account for cell-type specific expression within heterogeneous tissues

Reference demonstrates that ATP8A1 expression varies across tissues, with specific distribution patterns in retina that differ from ATP8A2, highlighting the importance of tissue-specific analysis.

Human Protein Atlas resources (linked to Prestige Antibodies) provide valuable tissue expression data across multiple normal and cancer tissues .

How can ATP8A1 antibodies be used to study disease processes and potential therapeutic targets?

ATP8A1 antibodies offer valuable tools for disease-related research:

• Cancer research applications:

  • Analyze ATP8A1 expression across tumor types and stages

  • Correlate expression with clinical outcomes and treatment responses

  • Investigate altered phospholipid asymmetry in cancer cells

  • Human Protein Atlas includes ATP8A1 analysis across common cancer types

• Neurological disorders:

  • Given ATP8A1's expression in brain tissue , investigate its role in:

    • Neurodegeneration

    • Synaptic function

    • Membrane remodeling during neurite outgrowth

  • Compare with ATP8A2, which has been linked to severe neurological disorders

• Liver diseases:

  • Examine potential cooperation between ATP8A1 and ATP8B1 (implicated in progressive familial intrahepatic cholestasis)

  • Analyze compensatory mechanisms in ATP8B1-deficient conditions

• Hematological applications:

  • Investigate ATP8A1's role in red blood cell (RBC) membrane asymmetry

  • Study implications for RBC disorders and hemoglobinopathies

  • Analyze PS exposure in various hematological conditions

• Therapeutic development:

  • Develop screening assays using ATP8A1 antibodies to identify modulators

  • Use antibodies to validate target engagement in drug development

  • Create companion diagnostics to identify patients likely to respond to therapies targeting phospholipid asymmetry

When designing these studies, consider that ATP8A1 works in complex with CDC50A and primarily localizes to endosomal compartments rather than the plasma membrane .

What are the current technical challenges in studying ATP8A1 and how might they be overcome?

Researchers face several challenges when studying ATP8A1:

• Distinguishing from other P4-ATPases:

  • Challenge: Structural similarity between ATP8A1 and related family members

  • Solutions:

    • Generate highly specific antibodies against unique epitopes

    • Use genetic approaches (CRISPR/siRNA) for specific targeting

    • Employ multiple detection methods to confirm findings

• Measuring flippase activity:

  • Challenge: Difficult to directly measure PS translocation in live cells

  • Solutions:

    • Develop improved fluorescent PS analogs with minimal perturbation

    • Use reconstituted systems with purified proteins

    • Combine biochemical assays (ATPase activity) with transport measurements

• Compartment-specific analysis:

  • Challenge: ATP8A1 functions in multiple endosomal compartments

  • Solutions:

    • Develop organelle-specific targeting of activity probes

    • Use subcellular fractionation combined with activity assays

    • Apply super-resolution microscopy for precise localization

• Handling membrane proteins:

  • Challenge: Purification and analysis of intact membrane proteins

  • Solutions:

    • Optimize detergent conditions for solubilization

    • Use nanodiscs or proteoliposomes for functional studies

    • Express protein in specialized systems (insect cells, yeast)

• Redundancy and compensation:

  • Challenge: Other P4-ATPases may compensate for ATP8A1 loss

  • Solutions:

    • Generate multiple knockout/knockdown models

    • Use acute inactivation approaches

    • Consider combined depletion of related flippases

Current approaches, such as those described in references , , and , provide methodological frameworks that can be adapted and improved to address these challenges.

How might emerging technologies enhance ATP8A1 research in the near future?

Emerging technologies will significantly advance ATP8A1 research:

• Single-cell analyses:

  • Single-cell proteomics to reveal cell-specific ATP8A1 expression patterns

  • Single-cell flippase activity assays to identify cellular heterogeneity

  • Integration with transcriptomics for comprehensive regulatory insights

• Advanced imaging approaches:

  • Super-resolution microscopy for precise subcellular localization

  • Live-cell FRET sensors to monitor ATP8A1 activity in real-time

  • Lattice light-sheet microscopy for dynamic 3D tracking of ATP8A1

  • Correlative light and electron microscopy for ultrastructural context

• Structural biology advancements:

  • Cryo-electron microscopy for high-resolution ATP8A1-CDC50A structures

  • Similar to recent structural studies of ATP8B1-CDC50A

  • Molecular dynamics simulations of lipid flipping mechanisms

  • Structure-guided antibody development targeting specific conformations

• Genome engineering approaches:

  • CRISPR base editing for introducing specific point mutations

  • Optogenetic or chemogenetic control of ATP8A1 activity

  • Endogenous tagging for visualization under physiological expression

  • Tissue-specific conditional knockout models

• Systems biology integration:

  • Multi-omics approaches linking ATP8A1 to broader cellular networks

  • Computational modeling of phospholipid asymmetry dynamics

  • Network analysis to identify new functional relationships

  • Machine learning for predicting ATP8A1 regulatory mechanisms

These technologies will enable researchers to address fundamental questions about ATP8A1 function and regulation with unprecedented precision and depth.