ATP2A2 Recombinant Monoclonal Antibody

What Is ATP2A2/SERCA2 and Why Is It an Important Research Target?

ATP2A2 (also known as SERCA2) is a magnesium-dependent enzyme that catalyzes the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to the sarcoplasmic/endoplasmic reticulum lumen. This protein plays critical roles in:

• Regulation of the contraction/relaxation cycle in muscle cells

• Calcium homeostasis in various cell types

• Autophagy regulation in response to starvation

• ER membrane contacts with other organelles including lipid droplets, mitochondria, and endosomes

ATP2A2 is particularly significant as mutations in this gene cause Darier-White disease (keratosis follicularis), an autosomal dominant skin disorder characterized by loss of adhesion between epidermal cells and abnormal keratinization .

The protein exists in multiple isoforms through alternative splicing, making it a complex target requiring specific antibodies for accurate detection in experimental contexts.

Technical Profile of ATP2A2:

What Applications Are ATP2A2 Recombinant Monoclonal Antibodies Validated For?

ATP2A2 recombinant monoclonal antibodies have been validated for multiple applications, though performance can vary between different clones and manufacturers. The main validated applications include:

• Western Blotting (WB): Most antibodies are validated at dilutions ranging from 1:500 to 1:20,000 depending on the specific antibody clone and manufacturer

• Immunohistochemistry (IHC): Typically used at dilutions of 1:100 to 1:1,000 for paraffin-embedded tissues

• Immunofluorescence/Immunocytochemistry (IF/ICC): Validated for cellular localization studies

• Flow Cytometry: Some clones are validated for intracellular staining

• Affinity Binding Assays: Certain antibodies have measured binding affinity (KD) values, such as 3.6 × 10⁻⁷ for some commercially available clones

When selecting an antibody for a specific application, researchers should review the validation data for each clone and consider the target epitope in relation to their experimental questions.

How Should Western Blot Protocols Be Optimized for ATP2A2 Detection?

Optimizing Western blot protocols for ATP2A2 detection requires consideration of several factors:

Sample Preparation and Loading:

• Use appropriate lysis buffers containing protease inhibitors to prevent degradation

• Load 20-30 μg of total protein per well, as demonstrated in validated protocols

• Include positive control samples (e.g., HEK-293 cells, A549 cells, skeletal muscle tissue)

Electrophoresis Conditions:

• Use 5-20% SDS-PAGE gels run at 70V (stacking)/90V (resolving) for 2-3 hours

• Expected molecular weight is approximately 110-120 kDa

Transfer and Detection:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

• Block with 5% milk in TBST for 1.5 hours at room temperature

• Incubate with primary antibody overnight at 4°C using manufacturer-recommended dilutions

• Wash three times with TBS-0.1% Tween for 5 minutes each

• Use appropriate HRP-conjugated secondary antibody (typically 1:1,000 dilution)

• Develop using enhanced chemiluminescence detection systems

Example Protocol from Validated Studies:

A validated Western blot protocol for ATP2A2 detection showed clear bands at 114 kDa using the following specific conditions:

• Primary antibody: 1:1,000 dilution overnight at 4°C

• Secondary antibody: Goat anti-rabbit IgG-HRP at 1:1,000 dilution for 1 hour at RT

• Development: Enhanced Chemiluminescent detection kit

How Do Different Epitope Targets Affect ATP2A2 Antibody Performance?

The epitope specificity of ATP2A2 antibodies significantly impacts their performance across different applications and experimental contexts:

N-Terminal vs. Middle Region Targeting:

• Antibodies targeting the N-terminal region (e.g., Sigma's clone 1G13) have shown robust performance in detecting ATP2A2 in various tissues and cell types with minimal background

• Middle region epitopes (amino acids 314-756, as in OTI4B12 clone) may offer different accessibility depending on protein conformation

Impact on Experimental Results:

• Epitope selection can affect detection of specific isoforms

• Conformational changes in ATP2A2 during its catalytic cycle (E1 vs. E2 states) may obscure certain epitopes

• Post-translational modifications, particularly oxidative stress-induced nitration on tyrosine residues 294 and 295, can affect antibody binding and may bias results when studying modified forms of the protein

Consideration for Different Applications:

The choice of epitope becomes particularly important when:

• Studying protein-protein interactions that might mask certain regions

• Investigating post-translational modifications

• Attempting to distinguish between different isoforms

• Examining conformational changes associated with calcium binding and release

For critical experiments, validating results with antibodies targeting different epitopes is recommended to ensure comprehensive detection.

What Controls Are Essential for ATP2A2 Antibody Validation?

Proper controls are crucial for validating ATP2A2 antibody specificity and performance:

Positive Controls:

• Cell lines: HEK-293, A549, HepG2, MCF-7, NIH/3T3, and C2C12 cells have been validated as expressing detectable levels of ATP2A2

• Tissues: Human skeletal muscle, cardiac muscle, and human urothelial tissue are reliable positive controls

Negative Controls:

• Knockout/Knockdown verification: ATP2A2 knockout or knockdown samples provide the most stringent negative controls

• Secondary antibody-only controls: Essential to confirm the absence of non-specific binding

• Blocking peptides: Competition assays using the immunizing peptide can confirm specificity

Cross-Reactivity Assessment:

• Testing across species (human, mouse, rat) is important if planning cross-species experiments

• The amino acid sequence conservation between species should be verified for the epitope region

• Some antibodies (e.g., OTI4B12 clone) have confirmed reactivity across human, mouse, and rat samples

Validation Methods:

• Western blot band specificity: Correct molecular weight (110-120 kDa)

• Immunohistochemistry pattern: Consistent with known expression patterns (e.g., pronounced expression in the subnuclear aspect of basal epidermal keratinocytes)

• Affinity measurements: KD values should be consistent with high-affinity binding (e.g., 3.6 × 10⁻⁷)

How Does UVB Irradiation Affect ATP2A2 Expression and Antibody Detection?

UVB irradiation has significant effects on ATP2A2 expression, which has important implications for experimental design and interpretation:

UVB-Induced Changes in ATP2A2:

• UVB irradiation reduces ATP2A2 mRNA levels in cultured normal keratinocytes

• This reduction may alter protein levels detectable by antibodies in exposed cells or tissues

• The effect appears to be mediated partly through inflammatory cytokines, particularly IL-6 and IL-8

Experimental Considerations:

• When studying UVB effects on skin or keratinocytes, researchers should account for potential reduction in ATP2A2 signal

• Time-course experiments may be necessary to capture the dynamic changes in expression

• Quantification should be normalized to appropriate housekeeping proteins that remain stable under UVB exposure

Therapeutic Interventions:

Research has shown that certain drugs can modulate the UVB-induced suppression of ATP2A2:

• Retinoids and corticosteroids inhibit UVB-induced suppression of ATP2A2 mRNA

• Anti-IL-6 antibodies prevent UVB-induced suppression

• Anti-IL-8 antibodies slightly accelerate the suppression

These findings have implications for designing experiments investigating ATP2A2 in the context of skin diseases like Darier's disease, where UVB exposure exacerbates symptoms.

What Are the Specific Challenges in Detecting ATP2A2 Isoforms?

ATP2A2 exists in multiple isoforms, presenting specific challenges for antibody-based detection:

Isoform Diversity:

• Multiple transcript variants arise from alternative splicing

• Isoform 2 is specifically involved in regulation of contraction/relaxation cycles and plays a role in TNFSF11-mediated Ca²⁺ signaling pathways

Antibody Selection Considerations:

• Epitope location is critical for isoform discrimination

• Some commercial antibodies may detect multiple isoforms if targeting conserved regions

• Researchers must verify whether the antibody of interest can distinguish between specific isoforms

Technical Approaches for Isoform Specificity:

• Epitope mapping: Select antibodies targeting isoform-specific regions

• Western blot analysis: Multiple bands may indicate detection of different isoforms

• RT-PCR validation: Complement antibody-based detection with transcript analysis

• Recombinant protein controls: Use purified isoforms as positive controls

Practical Example:

When investigating the immunogen sequence of commercial antibodies:

• Some target amino acids 314-756 of human ATP2A2 (NP_733765)

• Others target epitopes within 17 amino acids from the N-terminal region

Researchers must verify these regions against known isoform sequences to ensure appropriate detection of their isoform of interest.

How Can ATP2A2 Antibodies Be Used to Study Darier's Disease Mechanisms?

ATP2A2 antibodies are valuable tools for investigating the molecular mechanisms underlying Darier's Disease (DD):

Tissue Expression Patterns:

• In normal skin, ATP2A2 shows pronounced expression in the subnuclear aspect of basal epidermal keratinocytes with variable suprabasal expression

• ATP2A2 is also expressed in hair follicles (infundibulum and outer root sheath), sebaceous glands, eccrine glands, apocrine glands, and arrector pili muscle

• In Darier disease skin, strong ATP2A2 positivity is detected in basal, suprabasal, and acantholytic lesional cells

Experimental Approaches:

• Comparative IHC studies:

  • Compare expression patterns between normal and DD skin samples

  • Assess protein localization changes in lesional versus non-lesional areas

• Cell culture models:

  • Examine calcium dynamics in keratinocytes from DD patients

  • Study the effects of ATP2A2 mutations on protein function and localization

• Therapeutic intervention studies:

  • Evaluate how treatments like retinoids and corticosteroids affect ATP2A2 expression

  • Investigate the relationship between UVB exposure, cytokine production, and ATP2A2 expression

Research Findings:

Studies have shown that drugs effective for DD act by modulating ATP2A2 mRNA expression, suggesting potential therapeutic mechanisms . Additionally, the relationship between calcium signaling dysregulation and acantholysis (loss of cell-cell adhesion) in DD can be explored using appropriate antibodies to visualize ATP2A2 distribution and interactions with other proteins.

What Are the Optimal Storage and Handling Conditions for ATP2A2 Antibodies?

Proper storage and handling of ATP2A2 antibodies is crucial for maintaining their performance and extending their shelf life:

Storage Temperature Requirements:

• Most ATP2A2 antibodies should be stored at -20°C for long-term stability

• Some specialized formulations may require storage at -80°C

• Once thawed, aliquots for immediate use can be stored at 2-8°C for limited periods (typically 1-2 weeks)

Buffer Composition:

Common storage buffers include:

• PBS (pH 7.3) containing 1% BSA, 50% glycerol, and 0.02% sodium azide

• PBS with 0.02% sodium azide and 50% glycerol pH 7.3

• Some antibodies are available in BSA-free formulations for specialized applications

Handling Recommendations:

• Aliquoting: For antibodies stored at -20°C, aliquoting may be unnecessary, but is recommended for those requiring -80°C storage to avoid freeze-thaw cycles

• Thawing: Thaw on ice or at room temperature; avoid multiple freeze-thaw cycles

• Working dilutions: Prepare fresh working dilutions on the day of use

• Contamination prevention: Use sterile techniques to prevent microbial contamination

Stability Information:

• Most ATP2A2 antibodies remain stable for 12 months from the date of receipt when stored properly

• Avoid exposure to light for fluorophore-conjugated antibodies

• Check expiration dates and lot-specific information provided by manufacturers

How Does Oxidative Stress Affect ATP2A2 Detection and Function?

Oxidative stress has significant effects on ATP2A2 protein that can impact antibody detection and experimental interpretation:

Molecular Changes Under Oxidative Stress:

• ATP2A2 undergoes nitration on two specific tyrosine residues (amino acids 294 and 295) under oxidative stress conditions

• This post-translational modification reduces its catalytic activity

• These modifications can potentially alter epitope recognition by certain antibodies

Experimental Considerations:

• Epitope selection: Antibodies targeting regions containing tyrosines 294-295 may show differential binding to nitrated versus non-nitrated protein

• Functional studies: When studying ATP2A2 activity, oxidative conditions should be carefully controlled and reported

• Tissue/cell source: Samples from oxidative stress conditions (e.g., ischemia, aging, certain diseases) may show altered ATP2A2 detection patterns

Research Applications:

• Studying the relationship between oxidative stress and calcium dysregulation

• Investigating how nitration affects ATP2A2's role in autophagy and organelle contacts

• Exploring protective mechanisms against oxidative damage to calcium handling proteins

Relevant Research Findings:

Studies have demonstrated that the nitration-induced reduction in ATP2A2 catalytic activity contributes to calcium dysregulation in multiple pathological conditions, including heart failure and neurodegenerative diseases . Researchers investigating these conditions should consider how oxidative modifications might affect their antibody-based detection methods.

What Cross-Reactivity Considerations Exist When Using ATP2A2 Antibodies Across Species?

Cross-reactivity considerations are essential when planning experiments involving samples from different species:

Validated Species Reactivity:

• Many commercial ATP2A2 antibodies have confirmed reactivity with human, mouse, and rat samples

• Some antibodies may cross-react with other species based on epitope conservation, but formal validation is necessary

Sequence Homology Assessment:

The high degree of conservation in ATP2A2 across mammals facilitates cross-reactivity, but researchers should:

• Compare the antibody's epitope sequence across target species

• Request sequence alignment data from manufacturers when available

• Perform preliminary validation in new species before conducting full experiments

Testing in Non-Validated Species:

When testing ATP2A2 antibodies in species not validated by the manufacturer:

• Begin with Western blot to confirm the correct molecular weight

• Use positive control tissues (e.g., skeletal or cardiac muscle) known to express high levels of ATP2A2

• Consider using multiple antibodies targeting different epitopes for confirmation

Example of Cross-Reactivity Questions:

One customer inquiry documented in the search results asked about using an antibody validated for human, mouse, and rat on feline tissues . The manufacturer noted that while not specifically tested, there was a good chance of cross-reactivity based on sequence conservation, and offered an innovator award program for testing and validating this cross-reactivity.

How Can ATP2A2 Antibodies Be Used in Multiplex Imaging Applications?

ATP2A2 antibodies can be valuable tools in multiplex imaging studies to investigate calcium transport mechanisms in relation to other cellular components:

Selection Considerations for Multiplex Imaging:

• Antibody host species: Choose primary antibodies from different host species to avoid cross-reactivity of secondary antibodies

• Conjugation compatibility: For directly conjugated antibodies, select fluorophores with minimal spectral overlap

• Unconjugated formats: Some ATP2A2 antibodies are available in conjugation-ready formats (e.g., BSA and azide-free) for custom labeling

Optimal Co-localization Markers:

For studying ATP2A2's roles in different cellular contexts, consider co-staining with markers for:

• ER/SR membranes (e.g., calreticulin, PDI)

• Autophagy components (e.g., VMP1, LC3)

• Calcium signaling proteins (e.g., IP3R, RyR)

• Membrane contact site proteins for studying ATP2A2's role in organelle interactions

Protocol Optimization:

• Perform sequential staining if antibody cross-reactivity is a concern

• Optimize fixation methods (typically PFA for preserving membrane structures)

• Consider antigen retrieval requirements (TE buffer pH 9.0 or citrate buffer pH 6.0 have been validated)

Advanced Applications:

Recent developments in conjugation-ready antibody formats enable ATP2A2 detection in:

• Multi-parameter flow cytometry

• Mass cytometry (CyTOF)

• Multiplex immunofluorescence imaging

• Proximity ligation assays to study protein-protein interactions

What Are the Key Differences Between Conventional and Recombinant Monoclonal ATP2A2 Antibodies?

Understanding the differences between conventional and recombinant monoclonal antibodies targeting ATP2A2 is crucial for experimental design:

Production Methods:

• Conventional monoclonals: Produced by mouse hybridoma cells with potential batch-to-batch variation

• Recombinant monoclonals: Generated using recombinant DNA technology in defined expression systems (e.g., HEK293 cells)

Performance Characteristics:

Practical Advantages of Recombinant Antibodies:

• ZooMAb® recombinant antibodies offer "significantly enhanced specificity, affinity, reproducibility, and stability over conventional monoclonals"

• Recombinant technology allows precise engineering of the antibody sequence

• Production in defined expression systems eliminates contamination with animal proteins

Application Considerations:

For critical research applications, recombinant monoclonal antibodies may be preferred, particularly when:

• Long-term reproducibility is essential (e.g., biomarker studies)

• High specificity is required to distinguish between closely related proteins

• Batch-to-batch consistency is crucial for quantitative analyses

How Do ATP2A2 Expression Levels Vary Across Tissue Types and What Are the Implications for Antibody Dilution?

ATP2A2 expression varies significantly across tissue types, requiring optimization of antibody dilutions for each application:

Tissue Expression Patterns:

• Highest expression in cardiac and skeletal muscle

• Moderate expression in epithelial tissues including skin

• Variable expression in different regions of the nervous system

• Basal expression in most cell types for basic calcium homeostasis

Recommended Dilution Ranges by Application and Tissue:

Optimization Strategies:

• Begin with the manufacturer's recommended dilution

• Perform titration experiments to determine optimal signal-to-noise ratio

• Adjust based on the expression level in your specific tissue/cell type

• Consider enhanced detection methods for tissues with low expression

What Role Does ATP2A2 Play in Autophagy and How Can Antibodies Help Elucidate This Function?

ATP2A2 has recently been identified as a key player in autophagy regulation, opening new avenues for research using specific antibodies:

ATP2A2's Role in Autophagy:

• Involved in autophagy in response to starvation

• Upon interaction with VMP1 and activation, controls ER-isolation membrane contacts for autophagosome formation

• Modulates ER contacts with lipid droplets, mitochondria, and endosomes

Research Applications of ATP2A2 Antibodies:

• Co-localization studies: Visualize ATP2A2 positioning during autophagosome formation

• Protein-protein interaction analysis: Investigate ATP2A2's interactions with autophagy machinery

• Expression level monitoring: Assess how ATP2A2 levels change during autophagy induction

• Phosphorylation status: Examine regulatory modifications during autophagy

Experimental Approaches:

• Immunoprecipitation: Isolate ATP2A2 complexes to identify binding partners

• Proximity ligation assays: Visualize interactions between ATP2A2 and autophagy proteins

• Super-resolution microscopy: Examine the precise localization of ATP2A2 at membrane contact sites

• Time-course studies: Track ATP2A2 dynamics during autophagy progression

Emerging Research:

Recent studies have shown that ATP2A2's role extends beyond calcium pumping to structural functions at membrane contact sites. Using specific antibodies, researchers have demonstrated that ATP2A2 works in coordination with other proteins to mediate switching between ATP synthesis and thermogenesis, highlighting its multifunctional nature beyond calcium transport .