ATP2A3 Antibody

What is ATP2A3 and what biological functions does it perform?

ATP2A3 (SERCA3) is a magnesium-dependent enzyme that catalyzes the hydrolysis of ATP coupled with the transport of calcium from the cytosol into the sarcoplasmic/endoplasmic reticulum lumen. This protein contributes significantly to calcium sequestration involved in muscular excitation and contraction processes. The protein has a calculated molecular weight of approximately 109-114 kDa . ATP2A3 plays a crucial role in calcium signaling pathways and has been implicated in Alzheimer's disease pathways . The protein functions as an intracellular pump located in the sarcoplasmic or endoplasmic reticula of muscle cells, where it regulates calcium homeostasis essential for proper cellular function.

What types of ATP2A3 antibodies are commercially available for research?

Based on current research tools, ATP2A3 antibodies are available in several formats:

These antibodies are rigorously validated through various techniques including Western blot, immunohistochemistry, and immunofluorescence to ensure specificity and reproducibility in experimental applications .

What are the recommended storage conditions for ATP2A3 antibodies to maintain optimal activity?

For optimal antibody performance and longevity, ATP2A3 antibodies should generally be stored as follows:

For short-term storage (up to 2 weeks), maintain refrigerated at 2-8°C . For long-term storage, store at -20°C in small aliquots to prevent freeze-thaw cycles that can degrade antibody quality and performance . Most manufacturers supply ATP2A3 antibodies in stabilizing buffers, such as PBS with 0.09% sodium azide and additives like 2% sucrose or glycerol solutions to maintain antibody integrity . It is crucial to avoid repeated freeze-thaw cycles as this significantly reduces antibody efficacy. When working with the antibody, it is advisable to remove only the amount needed for immediate experiments and keep the main stock frozen .

What are the validated applications for ATP2A3 antibodies?

ATP2A3 antibodies have been validated for multiple research applications with specific optimized protocols:

• Western Blotting (WB): Most ATP2A3 antibodies are validated for WB with recommended dilutions ranging from 1:2000 to 1:50-1:200 depending on the specific antibody. The expected band size is approximately 114 kDa.

• Immunohistochemistry (IHC-P): ATP2A3 antibodies have been validated for use on paraformaldehyde-fixed, paraffin-embedded tissue sections with typical working dilutions of 1:25 to 1:50-1:200 .

• Immunocytochemistry/Immunofluorescence (ICC-IF): Several ATP2A3 antibodies have been validated for cellular localization studies through immunofluorescence techniques .

• ELISA: Some monoclonal antibodies against ATP2A3 demonstrate high sensitivity in ELISA applications, with detection limits for recombinant GST-tagged ATP2A3 reported at approximately 0.3 ng/ml .

Validation testing typically includes positive controls such as HL-60, Jurkat, and K562 cell lines, which express detectable levels of ATP2A3 protein .

How can I optimize ATP2A3 antibody-based immunohistochemistry to minimize background and maximize specific staining?

Optimizing immunohistochemistry with ATP2A3 antibodies requires a systematic approach to reduce background while enhancing specific signal detection:

What are the best techniques for quantifying ATP2A3 protein expression levels across different tissue samples?

Accurate quantification of ATP2A3 expression requires selecting appropriate techniques based on research objectives:

• Western Blotting with Densitometry: For semi-quantitative analysis, Western blotting using standardized lysate loading (20 μg per lane ) with normalization to housekeeping proteins provides reliable comparative data. Densitometric analysis should include standard curves with recombinant ATP2A3 protein for absolute quantification.

• Quantitative Immunohistochemistry: For tissue-specific expression patterns, quantitative IHC combining the validated dilutions (1:25-1:200 ) with digital image analysis offers spatial information alongside expression levels. Automated systems measuring staining intensity and distribution patterns should be calibrated using control tissues with known expression levels.

• ELISA-Based Quantification: For highly sensitive detection, sandwich ELISA using monoclonal capture antibodies with detection limits around 0.3 ng/ml provides precise quantification of soluble ATP2A3 in protein extracts.

• Multiplexed Protein Assays: For contextual analysis of ATP2A3 within signaling networks, multiplexed approaches combining ATP2A3 detection with other calcium signaling proteins provides comprehensive pathway activity assessment.

• Mass Spectrometry: For absolute quantification without antibody dependence, targeted proteomic approaches can quantify ATP2A3 peptides with high specificity and sensitivity, particularly valuable when comparing multiple SERCA isoforms simultaneously.

Each approach should incorporate appropriate reference standards and statistical analysis methods to ensure reliable cross-sample comparisons.

How do I troubleshoot non-specific binding when using ATP2A3 antibodies in Western blotting?

Non-specific binding in Western blots can significantly complicate ATP2A3 detection and quantification. A systematic troubleshooting approach includes:

• Antibody Validation: Confirm antibody specificity using multiple cell lines with known ATP2A3 expression. Validated positive controls include HL-60, Jurkat, and K562 cell lines . Consider using ATP2A3 knockdown or knockout samples as negative controls.

• Blocking Optimization: Standard 5% non-fat dry milk in TBST has been validated for ATP2A3 Western blots , but persistent non-specific binding may require alternative blocking agents such as 5% BSA or commercial blocking solutions.

• Antibody Dilution Adjustment: Begin with the manufacturer's recommended dilution (e.g., 1:2000 ) but systematically test higher dilutions if non-specific bands persist. Extending primary antibody incubation time while increasing dilution can maintain specific signals while reducing background.

• Stringent Washing: Implement additional washing steps with higher detergent concentrations (up to 0.1% Tween-20) in TBS buffer to remove weakly bound antibodies causing non-specific signals.

• Membrane Optimization: PVDF membranes may exhibit different non-specific binding profiles compared to nitrocellulose. Testing both membrane types can identify the optimal platform for ATP2A3 detection.

• Sample Preparation Refinement: ATP2A3 is a transmembrane protein requiring effective solubilization. Ensure complete denaturation by heating samples to 95°C for 5 minutes in SDS-PAGE loading buffer containing reducing agents before gel loading.

• Cross-Reactivity Assessment: If persistent bands appear at unexpected molecular weights, consider potential cross-reactivity with other SERCA family members or splice variants. Peptide competition assays can confirm antibody specificity.

What experimental controls should be incorporated when studying ATP2A3 expression in different cell types?

Robust experimental design for ATP2A3 expression studies requires comprehensive controls:

• Positive Expression Controls: Include cell lines with confirmed ATP2A3 expression such as HL-60, Jurkat, and K562 in all experiments to validate detection methods and establish expression baseline.

• Negative Controls:

  • Technical negatives: Include secondary antibody-only controls to assess non-specific binding

  • Biological negatives: Where available, incorporate ATP2A3 knockout or knockdown samples

• Antibody Validation Controls:

  • Peptide competition assays: Pre-incubate antibody with immunizing peptide to confirm binding specificity

  • Multiple antibody validation: Use two antibodies targeting different ATP2A3 epitopes (N-terminal and middle region ) to confirm expression patterns

• Normalization Controls:

  • For Western blots: House-keeping proteins appropriate to the cellular compartment (ER membrane proteins preferred over cytosolic proteins)

  • For qPCR: Multiple reference genes validated for stability in the specific experimental conditions

• Treatment/Condition Controls:

  • Vehicle controls for all treatments affecting calcium signaling

  • Time-matched controls for kinetic studies of ATP2A3 expression changes

• Cross-Isoform Controls: Include detection of other SERCA family members (SERCA1, SERCA2) to understand isoform-specific regulation and potential compensatory mechanisms.

• Subcellular Localization Controls: When performing immunofluorescence, include co-staining with established ER/SR markers to confirm proper subcellular localization of ATP2A3 signals.

How can I design experiments to investigate ATP2A3's role in calcium signaling pathways?

Designing experiments to elucidate ATP2A3's specific contributions to calcium signaling requires multifaceted approaches:

• Expression Modulation Strategies:

  • Implement CRISPR-Cas9 mediated knockout of ATP2A3 while monitoring calcium fluctuations with fluorescent indicators

  • Design isoform-specific siRNA knockdown experiments comparing effects of ATP2A3 silencing versus other SERCA family members

  • Create stable overexpression systems with tagged ATP2A3 constructs to assess dose-dependent effects on calcium dynamics

• Functional Activity Assessments:

  • Employ microsomal preparation techniques to isolate ER/SR-enriched fractions for direct measurement of ATP2A3-mediated calcium uptake kinetics

  • Use calcium ionophores and chelators in combination with real-time calcium imaging to assess ATP2A3 contribution to calcium store maintenance

  • Implement patch-clamp techniques in combination with ATP2A3 modulation to measure effects on store-operated calcium entry

• Pharmacological Manipulation:

  • Design dose-response experiments with thapsigargin (a known SERCA inhibitor) to compare ATP2A3 sensitivity versus other SERCA isoforms

  • Implement calcium pump activators in ATP2A3-expressing versus non-expressing cells to determine isoform-specific responses

• Pathway Integration Analysis:

  • Combine ATP2A3 modulation with stimulation of various calcium signaling pathways (e.g., IP3-mediated, ryanodine receptor-mediated) to determine pathway-specific contributions

  • Perform proteomic analysis of ATP2A3 interaction partners in different cell types to identify cell-specific regulatory mechanisms

• Disease-Relevant Models:

  • Design experiments in Alzheimer's disease models to assess ATP2A3's contribution to calcium dysregulation, as ATP2A3 has been implicated in Alzheimer's disease pathways

  • Investigate ATP2A3 expression and function in muscle excitation/contraction coupling disorders to determine pathophysiological relevance

• Technological Approaches:

  • Implement optogenetic tools to achieve temporally precise modulation of calcium signaling while monitoring ATP2A3 activity

  • Utilize FRET-based biosensors positioned at ER/SR membranes to directly monitor ATP2A3-mediated calcium transport in real-time

What are the key differences between polyclonal and monoclonal ATP2A3 antibodies for specific research applications?

Understanding the fundamental differences between polyclonal and monoclonal ATP2A3 antibodies is crucial for selecting the appropriate reagent:

For detecting native ATP2A3 in complex tissues, polyclonal antibodies often provide superior sensitivity due to recognition of multiple epitopes that remain accessible even after fixation and processing. This makes them particularly valuable for immunohistochemistry applications . Conversely, the consistent epitope recognition of monoclonal antibodies makes them ideal for quantitative applications where reproducibility is paramount, such as in ELISA-based detection systems with sensitivity reaching 0.3 ng/ml .

When designing critical experiments, consider using both antibody types as complementary approaches to strengthen research findings and confirm specificity of the observed ATP2A3 signals.

How can I properly validate and characterize a new ATP2A3 antibody for my research?

Comprehensive validation of a new ATP2A3 antibody requires a systematic multi-step approach:

• Epitope Analysis and Sequence Verification:

  • Confirm the immunogen sequence used (e.g., "synthetic peptide between 13-47 amino acids from human ATP2A3" or "synthetic peptide directed towards the middle region of human ATP2A3" )

  • Perform sequence homology analysis to assess potential cross-reactivity with other SERCA family members and across species

• Western Blot Validation:

  • Test against positive control lysates: HL-60, Jurkat, and K562 cell lines

  • Confirm band at expected molecular weight (114 kDa)

  • Perform peptide competition assay using the immunizing peptide

  • Include knockout/knockdown controls if available

• Immunoprecipitation Analysis:

  • Perform IP-Western to confirm ability to recognize native protein

  • Conduct reverse IP followed by mass spectrometry to identify all proteins pulled down

• Immunohistochemistry/Immunofluorescence Validation:

  • Test on known positive tissues (human brain has been validated )

  • Optimize fixation conditions (paraformaldehyde fixation has been validated )

  • Compare staining pattern with existing literature and other validated ATP2A3 antibodies

  • Perform co-localization studies with ER/SR markers to confirm expected subcellular distribution

• Specificity Testing:

  • Test reactivity against recombinant ATP2A3 protein and related SERCA isoforms

  • Assess cross-reactivity across species using the predicted sequence homology information

  • For human ATP2A3 antibodies, sequence homology analysis shows varying cross-reactivity: Dog (86%), Horse (86%), Pig (79%), Rat (79%), Bovine (79%), Guinea pig (79%), Rabbit (77%)

• Application-Specific Optimization:

  • Determine optimal working dilutions for each application through systematic titration

  • Document optimal conditions for antigen retrieval in IHC (citrate buffer, pH 6.0 has been validated )

  • Establish blocking conditions (3% BSA for 30 minutes at room temperature has been validated )

• Reproducibility Assessment:

  • Test multiple antibody lots if available

  • Compare results across different experimental conditions and biological samples

What factors impact the reproducibility of ATP2A3 antibody-based experiments?

Ensuring reproducible results with ATP2A3 antibodies requires careful consideration of multiple experimental variables:

• Antibody-Related Factors:

  • Lot-to-lot variability: Particularly significant for polyclonal antibodies ; maintain records of antibody lot numbers for all experiments

  • Storage conditions: Improper storage leading to antibody degradation; strictly adhere to manufacturer recommendations for temperature (-20°C for long-term ) and aliquoting to avoid freeze-thaw cycles

  • Age and stability: Antibody efficacy may decrease over time even with proper storage; validate older antibodies against fresh lots periodically

• Sample Preparation Factors:

  • Fixation methods: For IHC/IF, fixation duration and conditions significantly impact epitope accessibility; standardize fixation protocols (formaldehyde fixation has been validated )

  • Protein extraction efficiency: ATP2A3 is a membrane protein requiring effective solubilization; use consistent extraction buffers and protocols

  • Protein degradation: Include protease inhibitors in all extraction steps to prevent variable ATP2A3 degradation

• Technical Execution Factors:

  • Antigen retrieval methods: For IHC, standardize heat-mediated antigen retrieval with citrate buffer (pH 6.0)

  • Blocking conditions: Maintain consistent blocking solutions (3% BSA has been validated ) and durations

  • Antibody incubation parameters: Standardize dilutions (e.g., 1:2000 for WB ), temperature (37°C has been used ), and duration (1 hour has been validated )

  • Detection systems: Maintain consistent secondary antibodies and detection chemistries

• Quantification Factors:

  • Image acquisition settings: For microscopy-based applications, standardize exposure settings, gain, and objectives

  • Analysis algorithms: For quantitative applications, use consistent analysis pipelines and thresholding methods

  • Normalization approach: Select appropriate loading controls and normalization methods consistently

• Biological Variation Management:

  • Cell culture conditions: Cell density, passage number, and growth conditions can alter ATP2A3 expression

  • Tissue heterogeneity: In complex tissues, cellular composition variability can affect apparent ATP2A3 levels

  • Calcium signaling state: ATP2A3 function and potentially its detection may be influenced by cellular calcium levels and signaling activity

Implementing detailed standard operating procedures (SOPs) for all ATP2A3 antibody experiments and maintaining comprehensive records of all experimental conditions is essential for maximizing reproducibility.

How can ATP2A3 antibodies be effectively used to study cellular calcium homeostasis mechanisms?

ATP2A3/SERCA3 plays a crucial role in maintaining calcium homeostasis through its function as a calcium pump. To effectively use ATP2A3 antibodies in studying these mechanisms:

• Subcellular Localization Studies:

  • Implement co-immunofluorescence with ATP2A3 antibodies (dilution 1:25-1:200 ) alongside markers for ER/SR, Golgi, and plasma membrane to map compartment-specific calcium regulation

  • Use super-resolution microscopy techniques to visualize ATP2A3 distribution within membrane microdomains and potential colocalization with other calcium channels and pumps

  • Perform time-course studies following calcium perturbations to track potential redistribution of ATP2A3

• Expression-Function Correlation Studies:

  • Combine ATP2A3 immunodetection with live-cell calcium imaging using fluorescent indicators (Fura-2, Fluo-4) to correlate expression levels with calcium transient characteristics

  • Implement FRET-based calcium sensors targeted to specific subcellular compartments to measure ATP2A3's contribution to organelle-specific calcium pools

  • Correlate ATP2A3 expression (as measured by calibrated immunofluorescence) with calcium uptake kinetics in microsomal preparations

• Compensatory Mechanism Investigation:

  • Use multiplexed immunodetection to simultaneously quantify ATP2A3 alongside other calcium transporters (PMCA, NCX, other SERCA isoforms) to identify coordinated regulation

  • Apply ATP2A3 antibodies in tissues/cells from disease models with calcium dysregulation to assess adaptive changes in expression and localization

  • Combine with phospho-specific antibodies against known regulatory sites on ATP2A3 to correlate activity modulation with expression levels

• Tissue-Specific Function Analysis:

  • Apply validated ATP2A3 antibodies (e.g., in human brain tissue ) to identify cell-type specific expression patterns that correlate with specialized calcium signaling functions

  • Implement multi-color immunohistochemistry to identify potential cell-cell interactions mediated by differential ATP2A3 expression

  • Compare ATP2A3 expression patterns in tissues with distinct calcium signaling requirements (e.g., smooth muscle, endothelial cells, immune cells)

• Pathophysiological Applications:

  • Use ATP2A3 antibodies to assess expression changes in disease models with known calcium homeostasis disruption, particularly those associated with Alzheimer's disease pathways

  • Implement proximity ligation assays to detect ATP2A3 interactions with regulatory proteins under normal and pathological conditions

What are the best practices for using ATP2A3 antibodies in translational research and biomarker studies?

Implementation of ATP2A3 antibodies in translational and biomarker research requires specific methodological considerations:

• Tissue Microarray (TMA) Applications:

  • Validate ATP2A3 antibodies specifically for TMA applications using known positive tissues (brain, tonsil )

  • Establish standardized immunohistochemistry protocols with precise antibody dilutions (1:25-1:200 ), incubation times, and detection systems

  • Implement automated staining platforms to ensure consistency across large sample cohorts

  • Develop standardized scoring systems for ATP2A3 expression that account for both intensity and distribution patterns

• Biofluid-Based Biomarker Development:

  • Validate sandwich ELISA systems using monoclonal ATP2A3 antibodies for detection of potential secreted/released ATP2A3 in patient samples

  • Establish sensitivity limits (reported as low as 0.3 ng/ml ) and dynamic range in relevant biological matrices

  • Perform spike-recovery experiments to validate quantification accuracy in complex biological fluids

  • Assess pre-analytical variables affecting ATP2A3 stability in clinical samples

• Clinical Correlation Studies:

  • Standardize tissue collection, processing, and storage procedures to minimize pre-analytical variables affecting ATP2A3 detection

  • Implement digital pathology approaches for objective quantification of ATP2A3 immunostaining

  • Design appropriate statistical approaches for correlating ATP2A3 expression with clinical parameters, accounting for potential confounding variables

  • Validate findings across independent patient cohorts using identical antibodies and protocols

• Therapeutic Target Validation:

  • Develop cell-based assays incorporating ATP2A3 antibodies to screen for compounds affecting expression or post-translational modifications

  • Implement immunohistochemistry protocols in preclinical models to assess pharmacodynamic effects of compounds targeting calcium signaling pathways

  • Evaluate ATP2A3 expression as a potential predictive biomarker for response to calcium pathway-targeting therapeutics

• Multi-omics Integration:

  • Correlate protein-level expression data from ATP2A3 antibody-based assays with transcriptomic and proteomic datasets

  • Implement systems biology approaches to position ATP2A3 expression changes within broader pathway alterations

  • Combine with calcium signaling functional readouts to develop integrated biomarker signatures

• Reproducibility Considerations:

  • Adhere to REMARK guidelines for biomarker studies involving ATP2A3 antibodies

  • Implement rigorous validation across multiple antibody lots and detection platforms

  • Document detailed methodological parameters to enable reproduction in different laboratory settings

How does ATP2A3 expression vary across different human tissues and what are the implications for research design?

Understanding the tissue-specific expression patterns of ATP2A3 is crucial for experimental design and interpretation:

• Tissue Expression Profiles:

  • ATP2A3/SERCA3 shows distinct tissue-specific expression patterns that must be considered when designing experiments and selecting appropriate controls

  • Validated expression has been documented in:

    • Hematopoietic cells: Particularly in HL-60, Jurkat, and K562 cell lines

    • Brain tissue: Validated through immunohistochemistry

    • Lymphoid tissues: Including tonsil, as validated through immunoperoxidase staining

• Experimental Design Implications:

  • Positive Control Selection: When validating ATP2A3 antibodies or studying expression changes, incorporate tissue-appropriate positive controls

    • For blood cell studies: HL-60, Jurkat, or K562 cells serve as validated controls

    • For neurological studies: Human brain tissue sections have been validated

    • For lymphoid tissue research: Tonsil sections provide reliable positive controls

  • Expression Level Considerations: ATP2A3 expression levels vary significantly across tissues, requiring:

    • Adjusted antibody dilutions based on target tissue (ranging from 1:25 for low-expression tissues to 1:200 for high-expression tissues)

    • Modified detection systems appropriate to expression level (standard detection for high-expression; amplified detection for low-expression)

    • Careful selection of exposure times and imaging parameters to avoid saturation in high-expressing tissues while maintaining sensitivity for low-expressing tissues

• Isoform Considerations:

  • Splice Variant Awareness: Alternative splicing results in multiple ATP2A3 transcript variants encoding different isoforms

  • Epitope Selection: Consider whether the antibody epitope (N-terminal , middle region ) is present in all relevant isoforms in the target tissue

  • Isoform Specificity: Design experiments to distinguish ATP2A3 from other SERCA family members that may be co-expressed in the same tissue

• Subcellular Distribution Patterns:

  • ATP2A3 primarily localizes to sarcoplasmic/endoplasmic reticulum membranes, but the specific ER subdomains may vary by tissue type

  • Experimental designs should incorporate appropriate subcellular markers to confirm expected localization patterns in each tissue context

  • Fixation and permeabilization protocols may require tissue-specific optimization to preserve both antigenicity and subcellular architecture

• Pathological State Considerations:

  • ATP2A3 expression may be altered in disease states, particularly in pathways related to calcium dysregulation and Alzheimer's disease

  • Comparison between normal and pathological tissues should include careful matching of tissue regions, fixation conditions, and processing methods

  • Include gradient analysis of expression changes in relation to disease progression markers

Understanding these tissue-specific variations is essential for proper experimental design, appropriate control selection, and accurate interpretation of ATP2A3 antibody-based research findings.

What emerging technologies might enhance ATP2A3 detection and functional analysis?

The landscape of protein detection and functional analysis is rapidly evolving, offering new opportunities for ATP2A3 research:

• Advanced Imaging Technologies:

  • Super-resolution microscopy: Techniques such as STORM, PALM, and STED microscopy can resolve ATP2A3 distribution within ER/SR subdomains at nanometer resolution, far exceeding conventional confocal microscopy limits

  • Expansion microscopy: Physical expansion of specimens can reveal ATP2A3 organization relative to other calcium handling proteins with standard microscopy equipment

  • Live-cell ATP2A3 imaging: Development of minimally disruptive tagging strategies (e.g., split-GFP, HaloTag) compatible with ATP2A3 function to monitor dynamics in living cells

• Next-Generation Antibody Technologies:

  • Recombinant antibody fragments: Single-chain variable fragments (scFvs) and nanobodies against ATP2A3 offering improved tissue penetration and reduced background

  • Bivalent antibody constructs: Engineered antibodies recognizing two distinct ATP2A3 epitopes simultaneously to enhance specificity

  • Conformation-specific antibodies: Designed to recognize ATP2A3 in specific functional states (e.g., calcium-bound versus unbound)

• Single-Cell Analysis Applications:

  • Single-cell proteomics: Mass cytometry (CyTOF) with ATP2A3 antibodies for high-dimensional analysis of expression relative to other calcium signaling proteins at single-cell resolution

  • Spatial transcriptomics integration: Correlation of ATP2A3 protein expression with spatial transcriptomic data to understand regulatory mechanisms

  • Multimodal single-cell analysis: Integrated measurement of ATP2A3 expression, calcium dynamics, and functional readouts in individual cells

• Functional Genomics Approaches:

  • CRISPR screening: Genome-wide or focused screens to identify regulators of ATP2A3 expression and function

  • CRISPR base editing: Precise modification of ATP2A3 regulatory elements to dissect expression control mechanisms

  • CRISPR activation/inhibition: Targeted modulation of ATP2A3 expression without altering the genomic sequence

• Biosensor Development:

  • Activity-based sensors: Development of fluorescent or bioluminescent reporters specifically monitoring ATP2A3 catalytic activity rather than just expression

  • Conformation-sensitive probes: Sensors detecting ATP2A3 structural changes during calcium transport cycle

  • Proximity-based assays: Implementation of split luciferase or FRET-based approaches to monitor ATP2A3 interactions with regulatory partners

These emerging technologies promise to advance ATP2A3 research beyond current limitations, offering unprecedented insights into expression patterns, regulatory mechanisms, and functional roles in calcium homeostasis and signaling pathways.