ATP1A2 Antibody

What is ATP1A2 and why is it significant in neurological research?

ATP1A2 encodes the alpha 2 subunit of the Na+/K+-ATPase pump, a membrane protein responsible for establishing and maintaining electrochemical gradients by actively transporting three sodium ions out of the cell while bringing two potassium ions into the cell against their concentration gradients. This integral membrane protein is particularly expressed in astrocytes in the central nervous system, as well as in skeletal muscle, heart, and vascular smooth muscle tissues .

ATP1A2 is a significant research target because mutations in this gene are associated with several neurological disorders, most notably familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and various forms of epilepsy. The protein is also linked to cases of transient cytotoxic edema, polymicrogyria, neuromuscular periodic paralysis disorders, and recurrent coma with fever . Understanding ATP1A2's function provides critical insights into neural excitability, ion transport mechanisms, and the pathophysiology of these disorders.

What are the common applications of ATP1A2 antibodies in neuroscience research?

ATP1A2 antibodies serve multiple purposes in neuroscience research:

• Western Blotting (WB): For detecting and quantifying ATP1A2 protein levels in tissue lysates, typically revealing bands at approximately 97-102 kDa. Some antibodies may detect a degradation product at ~65 kDa .

• Immunohistochemistry (IHC): For visualizing ATP1A2 distribution in tissue sections, particularly useful for studying expression patterns in different brain regions or in pathological specimens. Recommended dilutions range from 1:50-1:500 .

• Immunofluorescence (IF): For determining subcellular localization and co-localization with other proteins. Typical working dilutions are 1:200-1:800 .

• Immunoprecipitation (IP): For isolating ATP1A2 protein complexes to identify interaction partners or study post-translational modifications. Typically requires 0.5-4.0 μg antibody per 1-3 mg of protein lysate .

• Flow Cytometry: For analyzing ATP1A2 expression in specific cell populations, with recommended usage around 0.25 μg per 10^6 cells .

For optimal results, antigen retrieval with TE buffer pH 9.0 is often recommended for fixed tissue samples, although citrate buffer pH 6.0 can sometimes be used as an alternative .

How do I choose between monoclonal and polyclonal ATP1A2 antibodies?

The choice between monoclonal and polyclonal ATP1A2 antibodies depends on your specific research objectives:

Monoclonal ATP1A2 antibodies (e.g., clone EPR11896(B)):

• Offer high specificity to a single epitope

• Provide consistent lot-to-lot reproducibility

• Ideal for quantitative studies or when background is a concern

• Examples include the rabbit recombinant monoclonal antibody that works well with human, mouse, and rat samples

Polyclonal ATP1A2 antibodies (e.g., catalog numbers 16836-1-AP, 55179-1-AP):

• Recognize multiple epitopes on the ATP1A2 protein

• Generally provide higher sensitivity

• More tolerant to minor protein denaturation or modifications

• Available with different host species (typically rabbit) and varying reactivity profiles

Consider these factors when making your selection:

• Application specificity - some antibodies perform better in certain applications (WB, IHC, IP)

• Target species compatibility - verify the antibody has been validated in your species of interest

• Isoform specificity - determine whether you need to distinguish ATP1A2 from other alpha subunit isoforms

• Epitope location - antibodies may recognize different regions of the protein (internal, N-terminal, C-terminal)

Review validation data from manufacturers and published literature to guide your selection.

What strategies can I use to ensure ATP1A2 antibody specificity?

Ensuring antibody specificity is critical for reliable ATP1A2 research. Implement these validation strategies:

• Multiple antibody approach:

  • Use at least two different ATP1A2 antibodies recognizing distinct epitopes

  • Consistent staining patterns increase confidence in specificity

• Positive and negative controls:

  • Positive controls: Tissues with known high ATP1A2 expression (skeletal muscle, brain tissue, astrocytes)

  • Negative controls: Tissues with minimal expression or knockout/knockdown models

  • For immunohistochemistry, include secondary-only controls to assess background

• Molecular validation:

  • Verify that detected bands match expected molecular weight (~100 kDa)

  • Be aware that degradation products may appear at ~65 kDa

  • Test against recombinant ATP1A2 protein when available

• Cross-reactivity assessment:

  • Some antibodies recognize all isoforms of the alpha subunit while others are ATP1A2-specific

  • If isoform specificity is crucial, verify the antibody has been validated against ATP1A1 and ATP1A3

• Subcellular localization validation:

  • Confirm that staining patterns match known localization (plasma membrane, particularly in astrocytes in CNS)

  • ATP1A2 should primarily show membrane localization consistent with its function

What are the recommended protocols for using ATP1A2 antibodies in Western blotting?

For optimal Western blotting results with ATP1A2 antibodies, follow this methodological approach:

Sample preparation:

• Homogenize tissues in RIPA buffer containing protease inhibitors

• For membrane proteins like ATP1A2, adding 0.5-1% SDS can improve extraction

• Important note: Some samples show better results when unboiled rather than boiled

Gel electrophoresis:

• Use 8-10% polyacrylamide gels to resolve the ~100-112 kDa ATP1A2 protein

• Load 20-30 μg of total protein per lane (as used in validated protocols)

Transfer and blocking:

• Transfer to PVDF membrane (nitrocellulose also acceptable)

• Block with 5% non-fat milk in TBST for 1 hour at room temperature

Antibody incubation:

• Dilute primary ATP1A2 antibody as recommended (typically 1:500-1:2000)

• Incubate overnight at 4°C with gentle agitation

• Wash 3-5 times with TBST

• Incubate with appropriate HRP-conjugated secondary antibody (1:2000-1:5000)

Detection:

• Develop using ECL substrate

• Expected molecular weight is approximately 97-102 kDa

Optimization tips:

• Include positive control tissues (brain, skeletal muscle, heart tissue)

• For best results with mouse samples, brain tissues are particularly recommended

• Some antibodies detect degradation products (~65 kDa)

How can I optimize immunohistochemistry protocols for ATP1A2 detection?

Optimizing immunohistochemistry for ATP1A2 detection requires careful attention to several parameters:

Tissue preparation:

• Perfusion fixation with 4% paraformaldehyde provides better preservation of membrane proteins

• Post-fixation time should be optimized (4-24 hours) to balance antigen preservation and tissue integrity

• For paraffin sections, use thin sections (5 μm) to facilitate antibody penetration

Antigen retrieval:

• Heat-induced epitope retrieval is typically necessary

• Use TE buffer pH 9.0 as recommended for many ATP1A2 antibodies

• Citrate buffer pH 6.0 can be used as an alternative

Blocking and antibody incubation:

• Block with 5-10% normal serum (from the species of secondary antibody)

• Dilute ATP1A2 antibody appropriately (typically 1:50-1:500, depending on the antibody)

• Incubate at 4°C overnight in a humidity chamber

• For double-labeling studies, combine with cell-type specific markers (GFAP for astrocytes)

Detection system:

• For chromogenic detection, use a polymer-based detection system for increased sensitivity

• For fluorescence, select secondary antibodies with minimal cross-reactivity

• Include DAPI for nuclear counterstain

Controls:

• Include a no-primary antibody control on each slide

• Process ATP1A2-rich tissues (skeletal muscle, heart tissue) in parallel as positive controls

How can I differentiate between Na+/K+ ATPase alpha subunit isoforms using antibodies?

Differentiating between highly homologous Na+/K+ ATPase alpha subunit isoforms (ATP1A1, ATP1A2, ATP1A3) requires careful antibody selection and experimental design:

Antibody selection strategies:

• Choose isoform-specific antibodies targeting unique regions

• Verify that the antibody has been validated against all isoforms to demonstrate specificity

• ATP1A2-specific antibodies like 55179-1-AP are designed to recognize only the ATP1A2 isoform

• Some antibodies (e.g., H-3 clone) recognize all alpha isoforms and require additional methods for differentiation

Experimental approach for isoform differentiation:

• Tissue/cell selection based on differential expression:

  • ATP1A1: Ubiquitously expressed

  • ATP1A2: Predominant in astrocytes, skeletal muscle, and heart tissue

  • ATP1A3: Neuronal-specific

  • Compare staining patterns in these tissues to confirm specificity

• Western blot optimization:

  • Use high-resolution SDS-PAGE (6-8% gels) for optimal separation

  • Include positive control lysates for each isoform

  • Some antibodies can recognize all isoforms of alpha subunit

• Immunofluorescence co-localization:

  • Double-label with cell-type specific markers (neurons for ATP1A3, astrocytes for ATP1A2)

  • ATP1A2 should co-localize primarily with astrocyte markers in CNS tissues

How can I use ATP1A2 antibodies to study the role of this protein in migraine pathophysiology?

ATP1A2 mutations are strongly linked to familial hemiplegic migraine type 2 (FHM2), making it an important target for migraine research. Here's a methodological approach:

Genetic and protein analysis in patient samples:

• Mutation screening:

  • Screen for ATP1A2 mutations in patients with hemiplegic migraine

  • Both familial and sporadic cases should be considered

• Protein expression analysis:

  • Use ATP1A2 antibodies to compare protein expression between patient and control samples

  • Western blot analysis of patient-derived cells (fibroblasts or lymphoblasts)

  • Immunohistochemistry on available tissue samples

Functional studies in model systems:

• Expression of mutant ATP1A2 in cellular models:

  • Introduce identified mutations via site-directed mutagenesis

  • Express wild-type and mutant ATP1A2 in appropriate cell lines

  • Use ATP1A2 antibodies to assess:

    • Total protein expression levels

    • Subcellular localization (membrane vs. intracellular)

    • Stability and degradation rates

• Cortical spreading depression (CSD) models:

  • CSD is the physiological correlate of migraine aura

  • Use ATP1A2 antibodies to examine distribution and expression changes before, during, and after CSD

  • Combine with electrophysiology to correlate ATP1A2 expression with functional changes

Translational approaches:

• ATP1A2 in mouse models of migraine:

  • Knock-in mice carrying human FHM2 mutations

  • Use ATP1A2 antibodies for immunohistochemistry to analyze:

    • Regional distribution in brain tissues

    • Changes in expression following migraine triggers

    • Co-localization with astrocyte markers

• Therapeutic target validation:

  • Test compounds that modulate Na+/K+ ATPase function

  • Use ATP1A2 antibodies to assess target engagement

  • Correlate with behavioral and electrophysiological outcomes

What approaches are recommended for studying ATP1A2 in cardiovascular tissues?

ATP1A2 is expressed in heart and vascular smooth muscle, where it plays important roles in contractility and blood pressure regulation. A recent study even identified a potential link between ATP1A2 mutations and cardiac arrhythmias . Here's how to study ATP1A2 in cardiovascular contexts:

Tissue preparation and immunolabeling:

• For whole heart: Langendorff perfusion with fixative ensures better preservation

• For vessels: Pressure-fixation maintains physiological dimensions

• Use ATP1A2 antibodies at appropriate dilutions (1:50-1:500)

• Co-stain with cardiomyocyte markers (α-actinin) or vascular smooth muscle markers (α-SMA)

Functional correlation:

• ATP1A2 distribution in cardiac tissues:

  • The α2 subunit accounts for approximately 15% of total Na+/K+ ATPase content in cardiomyocytes

  • Use immunofluorescence to determine precise subcellular localization

  • Co-label with Na+/Ca2+ exchanger (NCX) to examine functional coupling

• ATP1A2 in cardiovascular disease models:

  • Compare ATP1A2 expression in normal vs. hypertrophic or failing hearts

  • Analyze ATP1A2 distribution in vascular tissues from hypertensive models

  • Assess relationship between ATP1A2 expression and arrhythmia susceptibility

Mechanistic studies:

• ATP1A2 regulates cardiomyocyte contractility by controlling intracellular Na+ concentration and consequently Ca2+ levels through the Na+/Ca2+ exchanger

• Altered Na+ and Ca2+ concentrations can lead to both atrial and ventricular arrhythmias

• Use ATP1A2 antibodies to track expression changes during disease progression

What are common challenges when using ATP1A2 antibodies and how can they be addressed?

Working with ATP1A2 antibodies presents several challenges that can be systematically addressed:

Challenge 1: Weak or absent signal in Western blots

• Solution: Optimize protein extraction for membrane proteins

  • Use stronger lysis buffers containing 1% SDS or 0.5% NP-40

  • Avoid boiling samples as this may cause aggregation of membrane proteins

  • Try 37°C incubation instead of boiling

  • Increase antibody concentration and/or extend incubation time

Challenge 2: High background in immunohistochemistry

• Solution: Optimize blocking and antibody conditions

  • Use stronger blocking (5-10% normal serum plus 1% BSA)

  • Include 0.1% Tween-20 in antibody diluent

  • Perform more extensive washing steps

  • Try a different antigen retrieval method (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

Challenge 3: Cross-reactivity with other alpha isoforms

• Solution: Verify isoform specificity

  • Some antibodies recognize all alpha isoforms

  • Use ATP1A2-specific antibodies when discrimination is critical

  • Include appropriate positive controls for each isoform

  • Complement protein detection with mRNA analysis (qPCR)

Challenge 4: Inconsistent results between applications

• Solution: Application-specific optimization

  • Antibodies that work well for WB may not be optimal for IHC and vice versa

  • Check manufacturer recommendations for each application

  • Conduct systematic titration for each application

  • Consider using different antibodies optimized for specific applications

Challenge 5: Poor reproducibility between experiments

• Solution: Standardize protocols and controls

  • Document lot numbers and create reference samples

  • Include positive controls (brain tissue, skeletal muscle) in each experiment

  • Maintain consistent sample preparation procedures

  • Use automated systems when possible to reduce variation

How should ATP1A2 antibodies be stored and handled to maintain their performance?

Proper storage and handling of ATP1A2 antibodies is essential for maintaining their performance over time:

Storage conditions:

• Store antibodies at -20°C for long-term storage

• Most ATP1A2 antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Antibodies are typically stable for one year after shipment when stored properly

• Aliquoting is generally unnecessary for -20°C storage but recommended if frequent use is anticipated

Handling recommendations:

• Thawing and preparation:

  • Thaw antibodies completely before use

  • Mix gently by inverting or gentle flicking (avoid vortexing)

  • Brief centrifugation to collect liquid at the bottom of the tube

  • Keep on ice when in use

• Working solution preparation:

  • Prepare fresh working dilutions on the day of use

  • Use high-quality, filtered buffers to prepare dilutions

  • For Western blot, dilute in 5% BSA or milk in TBST

  • For IHC/IF, dilute in recommended antibody diluent

• Contamination prevention:

  • Use sterile techniques when handling antibodies

  • Never return unused antibody to the original vial

  • Use clean pipette tips for each handling

  • Monitor for signs of contamination (cloudiness, precipitates)

Performance monitoring:

• Include internal controls in each experiment to monitor antibody performance

• Document lot numbers and testing dates

• Consider creating a reference sample set to check new lots

• If performance decreases, try fresh dilutions before troubleshooting other variables

How can ATP1A2 antibodies be used to study rare neurological disorders?

ATP1A2 mutations are associated with several rare neurological disorders, and antibodies can be valuable tools for studying these conditions:

Familial Hemiplegic Migraine Type 2 (FHM2):

• Use ATP1A2 antibodies to compare protein expression, stability, and localization between wild-type and mutant forms

• Immunofluorescence can reveal if FHM2 mutations affect membrane targeting

• Western blot analysis can determine if mutations alter protein stability or expression levels

Alternating Hemiplegia of Childhood (AHC):

• ATP1A2 mutations can cause AHC, a rare neurological disorder characterized by episodes of hemiplegia

• Use immunohistochemistry to analyze ATP1A2 distribution in available brain tissues

• Patient-derived cellular models can be analyzed with ATP1A2 antibodies to study functional defects

MELAS-like syndrome:

• A recent case study identified a novel ATP1A2 variant in a patient with MELAS-like alternating hemiplegia

• ATP1A2 antibodies can help characterize protein expression in this unusual phenotype

• Correlate ATP1A2 expression with MRI findings showing abnormal linear signals in the cerebral cortex

Methodological approach:

• Patient-derived models:

  • Generate induced pluripotent stem cells (iPSCs) from patient samples

  • Differentiate into relevant cell types (neurons, astrocytes)

  • Use ATP1A2 antibodies to characterize expression and localization

• CRISPR-engineered models:

  • Introduce specific ATP1A2 mutations using CRISPR/Cas9

  • Validate using sequencing

  • Compare protein characteristics with patient samples using ATP1A2 antibodies

• Functional correlation:

  • Correlate protein findings with clinical phenotypes (attack frequency, age of onset, etc.)

  • Establish genotype-phenotype correlations

  • Compare ATP1A2 function across different patient mutations

What is the relevance of ATP1A2 in cardiovascular disorders and how can antibodies help study this connection?

Recent research has revealed intriguing connections between ATP1A2 and cardiovascular disorders:

ATP1A2 in cardiac function:

• The α2 subunit accounts for approximately 15% of the total Na+/K+ ATPase content in cardiomyocytes

• The Na+/K+ ATPase pump regulates cardiomyocyte contractility by controlling intracellular Na+ concentration and consequently Ca2+ levels through the Na+/Ca2+ exchanger

• Altered Ca2+ and Na+ concentrations can lead to both atrial and ventricular arrhythmias

Clinical relevance:

• A recent study described co-occurrence of familial hemiplegic migraine and cardiac arrhythmias resistant to antiarrhythmic drugs in a patient with an ATP1A2 mutation

• This suggests a potential causal relationship between ATP1A2 mutations and heart arrhythmias

Research approaches using ATP1A2 antibodies:

• Expression analysis in cardiovascular tissues:

  • Use Western blotting with ATP1A2 antibodies to compare expression levels in normal vs. diseased heart tissues

  • Immunohistochemistry to evaluate regional distribution in different chambers of the heart

• Cellular localization studies:

  • Immunofluorescence to determine subcellular localization in cardiomyocytes

  • Co-localization with ion channels and transporters relevant to arrhythmogenesis

  • Evaluation of potential redistribution during disease processes

• Mechanistic investigations:

  • Determine if ATP1A2 mutations alter protein-protein interactions in cardiac tissue

  • Assess functional coupling with the Na+/Ca2+ exchanger

  • Evaluate effects on calcium handling and action potential characteristics

• Translational potential:

  • Test if ATP1A2 expression correlates with arrhythmia susceptibility

  • Evaluate ATP1A2 as a potential biomarker for specific cardiac pathologies

  • Investigate whether targeting ATP1A2 might have therapeutic applications