ATP2B4 Antibody

What is ATP2B4 and why is it significant in scientific research?

ATP2B4 (ATPase plasma membrane Ca2+ transporting 4) is a critical membrane protein involved in calcium homeostasis and signaling. In humans, the canonical protein comprises 1241 amino acid residues with a molecular mass of approximately 137.9 kDa. Its primary subcellular localization is in the cell membrane, where it functions as a calcium pump. ATP2B4 belongs to the Cation transport ATPase (P-type) (TC 3.A.3) protein family and plays vital roles in brain development and intracellular calcium regulation. The protein's significance in research stems from its involvement in calcium signaling pathways, which are fundamental to numerous physiological processes including muscle contraction, neural transmission, and hormone secretion .

How do ATP2B4 antibodies differ from other calcium transporter antibodies?

ATP2B4 antibodies are specifically designed to target the unique epitopes of ATP2B4 (also known as PMCA4), distinguishing it from other calcium transporters such as ATP2B1 (PMCA1), ATP2B2 (PMCA2), and ATP2B3 (PMCA3). While these calcium transporters share structural similarities as members of the P-type ATPase family, ATP2B4 antibodies typically target unique regions, including the C-terminal domain or middle regions that differ significantly between PMCA isoforms. This specificity is crucial for accurate identification and characterization of ATP2B4's distinct roles in calcium homeostasis compared to other calcium transporters . When selecting an antibody, researchers should consider the specific isoform (among the 8 reported alternative splice variants of ATP2B4) they wish to detect, as some antibodies may recognize multiple splice variants while others target specific forms.

What are the common synonyms and alternative names for ATP2B4 that researchers should be aware of?

When searching literature or ordering antibodies, researchers should be aware that ATP2B4 is known by several alternative names and identifiers, including MXRA1, PMCA4, PMCA4b, PMCA4x, and plasma membrane calcium-transporting ATPase 4. Occasionally, it may be incorrectly labeled as ATP2B2 in some databases or resources . This nomenclature diversity reflects the protein's discovery history and functional characterization by different research groups. When conducting literature searches or selecting antibodies, using multiple search terms encompassing these synonyms ensures comprehensive coverage of relevant research and available reagents.

What applications are ATP2B4 antibodies most commonly used for in research?

ATP2B4 antibodies are utilized across multiple experimental applications, with the most common being Western Blot (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunocytochemistry (ICC), and Flow Cytometry (FCM). Most commercially available antibodies are validated for multiple applications, though performance may vary between applications. For instance, the JA9 clone has been extensively validated for Western Blot, ELISA, FCM, ICC, IF, IHC-frozen sections, IHC-paraffin sections, and Immunoprecipitation (IP), making it one of the most versatile antibodies for ATP2B4 research . When designing experiments, researchers should select antibodies that have been specifically validated for their intended application rather than assuming cross-application functionality.

How should researchers optimize antibody dilutions for Western blot detection of ATP2B4?

Optimizing antibody dilutions for Western blot detection of ATP2B4 requires systematic titration to balance signal intensity with background. For primary antibodies, start with the manufacturer's recommended dilution (typically 1:500 to 1:2000) and perform a dilution series (e.g., 1:500, 1:1000, 1:2000, 1:5000) using a constant sample with known ATP2B4 expression. The large size of ATP2B4 (137.9 kDa) requires efficient protein transfer, so extended transfer times or specialized transfer conditions for high molecular weight proteins should be considered . Additionally, researchers should optimize blocking conditions, as ATP2B4 detection can be sensitive to blocking reagent composition. For quantitative Western blots, always include appropriate loading controls and standardize protein quantities across samples. Document optimal conditions for reproducibility in future experiments.

What are the key considerations when selecting between monoclonal and polyclonal ATP2B4 antibodies?

When selecting between monoclonal and polyclonal ATP2B4 antibodies, researchers should consider several factors based on their experimental needs:

For critical experiments, validation using both antibody types or multiple antibodies targeting different epitopes provides stronger evidence for observations. When studying specific ATP2B4 isoforms, select antibodies raised against regions that can differentiate between the eight reported splice variants .

How can ATP2B4 antibodies be applied in studying calcium signaling dysregulation in disease models?

ATP2B4 antibodies serve as powerful tools for investigating calcium signaling dysregulation in disease models through multiple methodological approaches. In primary aldosteronism research, ATP2B4 antibodies have been used to examine how ATP2B4 variants affect calcium regulation in adrenal cell models, providing insights into mechanisms of hormone production disregulation . For experimental design, researchers should employ a multi-faceted approach: (1) Quantitative immunoblotting to assess ATP2B4 expression levels across disease and control samples; (2) Immunolocalization to determine if subcellular distribution changes in pathological states; (3) Co-immunoprecipitation coupled with mass spectrometry to identify disease-specific interaction partners; and (4) Proximity ligation assays to confirm altered protein-protein interactions in situ. This comprehensive approach enables correlation of ATP2B4 dysfunction with downstream calcium-dependent signaling events, particularly in cellular models of hypertension, neurological disorders, and cardiovascular diseases where calcium homeostasis plays crucial roles.

What strategies should be employed when using ATP2B4 antibodies in gene variant functional studies?

When using ATP2B4 antibodies in gene variant functional studies, researchers should implement a systematic approach that integrates multiple techniques. Start by developing cellular models expressing wild-type and variant ATP2B4 forms using inducible expression systems (as demonstrated with HAC15-dox-ATP2B4 cells) . Verify variant expression using antibodies that recognize regions distant from the mutation site to ensure detection regardless of structural changes. Implement the following experimental design:

• Quantitative immunoblotting to compare expression levels between wild-type and variant proteins

• Immunofluorescence to assess changes in subcellular localization

• Functional calcium imaging coupled with immunocytochemistry to correlate protein expression with calcium transport activity

• Electrophysiological measurements in antibody-verified cells to directly assess functional differences

When analyzing ATP2B4 variants associated with diseases like primary aldosteronism, compare antibody-based protein detection with parallel functional readouts such as luciferase reporter assays (e.g., HAC15-B2Luc cells) to link protein expression with downstream functional consequences . Document any discrepancies between transcript and protein levels, as these may provide insights into variant-specific post-transcriptional regulation.

How can ATP2B4 antibodies be utilized in the study of protein-protein interactions involving calcium transport systems?

ATP2B4 antibodies provide sophisticated tools for investigating protein-protein interactions in calcium transport systems through multiple methodological approaches. For comprehensive interaction studies, implement a multi-stage experimental strategy:

• Co-immunoprecipitation (Co-IP): Use ATP2B4 antibodies validated for IP applications (such as the JA9 clone) to pull down ATP2B4 complexes from cellular lysates under non-denaturing conditions, followed by mass spectrometry or immunoblotting to identify interaction partners .

• Proximity Ligation Assays (PLA): Combine ATP2B4 antibodies with antibodies against suspected interaction partners to visualize and quantify interactions in situ with single-molecule sensitivity.

• FRET-based approaches: Use ATP2B4 antibody fragments conjugated with fluorophores as FRET donors/acceptors to study dynamic interactions in living cells.

• Bimolecular Fluorescence Complementation (BiFC): Combine with genetic approaches for validation of interactions identified through antibody-based methods.

When investigating interactions between ATP2B4 and proteins involved in calcium signaling networks or regulatory complexes, careful validation of antibody specificity is essential to avoid false positives due to cross-reactivity with other PMCA isoforms. Document interaction dependencies on calcium levels by conducting experiments in buffer systems with varying calcium concentrations to capture dynamic, calcium-dependent interactions.

What are the optimal tissue preparation techniques for immunohistochemical detection of ATP2B4?

When working with brain tissue, where ATP2B4 plays crucial roles in development and calcium regulation, perfusion fixation improves antigen preservation and reduces background. For dual immunofluorescence studies examining ATP2B4 co-localization with other membrane proteins, mild fixation protocols (0.5-2% paraformaldehyde) better preserve membrane integrity. Always include positive control tissues with known ATP2B4 expression (e.g., brain, cardiac tissue) and negative controls omitting primary antibody. When optimizing new antibodies or tissues, prepare a test panel using multiple fixation protocols and epitope retrieval methods to determine optimal conditions for specific experimental needs.

How should researchers approach ATP2B4 knockdown validation using antibodies?

Validating ATP2B4 knockdown requires a methodical approach combining antibody-based detection with functional analyses. When using short hairpin RNA (shRNA) or siRNA targeting ATP2B4, verification should employ multiple techniques:

• Western blot validation: Quantify protein reduction using antibodies targeting regions outside the knockdown-targeted sequence. Calculate knockdown efficiency through densitometric analysis normalized to loading controls (e.g., GAPDH, β-actin) .

• Immunofluorescence confirmation: Visualize reduction in ATP2B4 signal intensity through immunofluorescence microscopy, with quantitative image analysis of signal intensity across multiple fields and experiments.

• Functional validation: Correlate antibody-detected protein reduction with functional readouts such as calcium flux measurements or reporter systems (e.g., CYP11B2-driven luciferase in HAC15-B2Luc cells for aldosterone production studies) .

• Rescue experiments: Perform rescue experiments with shRNA/siRNA-resistant ATP2B4 constructs to confirm phenotype specificity.

When interpreting knockdown results, consider the stability of the ATP2B4 protein (which may result in delayed protein reduction relative to mRNA knockdown) and potential compensatory upregulation of other PMCA isoforms. Document both the percentage reduction in protein levels and corresponding functional consequences to establish clear knockdown-phenotype relationships.

What protocols should be followed for successful immunoprecipitation of ATP2B4?

Successful immunoprecipitation of ATP2B4 requires careful optimization of lysis conditions, antibody selection, and experimental protocols. The following methodology ensures effective isolation of ATP2B4 and its complexes:

• Cell/tissue lysis optimization: Use mild, non-ionic detergent buffers (e.g., 1% NP-40 or 0.5% Triton X-100) in Tris-buffered saline with protease inhibitors and 1-2 mM calcium chelators (EGTA/EDTA) to preserve protein interactions. For membrane proteins like ATP2B4, lysis buffer optimization is critical – test multiple detergent concentrations to balance protein solubilization with preservation of interactions .

• Antibody selection: Choose antibodies specifically validated for immunoprecipitation, such as the JA9 clone which has been documented for IP applications. Pre-clear lysates with protein A/G beads before adding antibodies to reduce non-specific binding .

• Binding conditions: Incubate antibodies with lysates overnight at 4°C with gentle rotation to maximize antigen capture while minimizing proteolytic degradation.

• Washing stringency gradient: Implement a gradient of washing stringency, using buffers with decreasing detergent concentrations to remove non-specific interactions while preserving specific ones.

For studying interacting partners, consider crosslinking approaches before lysis to capture transient interactions. When analyzing post-translational modifications of ATP2B4, include appropriate phosphatase inhibitors (for phosphorylation studies) or deubiquitinase inhibitors (for ubiquitination studies) in lysis buffers. Always validate IP efficiency through immunoblotting a small fraction of the immunoprecipitate with a different ATP2B4 antibody recognizing a separate epitope.

How can researchers resolve conflicting data when different ATP2B4 antibodies produce inconsistent results?

When facing conflicting results from different ATP2B4 antibodies, implement a systematic troubleshooting approach:

• Epitope mapping analysis: Determine the exact epitopes recognized by each antibody – discrepancies may arise when antibodies target different domains or isoform-specific regions of ATP2B4. The eight reported splice variants may be differentially detected depending on epitope location .

• Validation with genetic approaches: Confirm antibody specificity through parallel genetic techniques such as CRISPR/Cas9 knockout, siRNA knockdown, or overexpression systems. Compare antibody signals in these controlled genetic backgrounds to determine specificity .

• Cross-validation with multiple techniques: Test antibodies across multiple applications (WB, IF, IHC) to identify application-specific inconsistencies versus genuine detection discrepancies.

• Positive and negative controls: Include tissues/cells with known high (brain, heart) and low/absent ATP2B4 expression patterns to evaluate each antibody's sensitivity and specificity.

When reporting results, document all antibodies used, their clone numbers or catalog information, working dilutions, and specifically note any discrepancies observed between different antibodies. Consider the possibility that conflicting results may reflect biologically relevant phenomena such as post-translational modifications, protein-protein interactions, or conformational changes that differentially affect epitope accessibility.

What are the common pitfalls in quantitative analysis of ATP2B4 expression using antibody-based methods?

Quantitative analysis of ATP2B4 expression using antibody-based methods presents several challenges that require careful experimental design and data interpretation:

• Saturation effects: Traditional Western blot analysis can become non-linear at high protein loads or with excessive antibody concentration. Establish a standard curve with serial dilutions of samples to determine the linear detection range for accurate quantification .

• Isoform-specific quantification challenges: The presence of eight ATP2B4 splice variants complicates quantification if antibodies recognize multiple isoforms. Select antibodies targeting common regions for total ATP2B4 quantification or isoform-specific regions for variant-specific analysis .

• Reference protein selection: Membrane protein quantification is particularly sensitive to loading control selection. Validate multiple reference proteins (beyond standard GAPDH or β-actin) to identify those that remain stable under your experimental conditions.

• Subcellular redistribution misinterpretation: Changes in ATP2B4 localization can be misinterpreted as expression changes in certain techniques. Complement whole-cell quantification with subcellular fractionation or imaging approaches to distinguish between expression changes and redistribution effects.

For immunofluorescence quantification, implement standardized image acquisition parameters, analyze multiple fields per sample, and use automated analysis algorithms to reduce subjective bias. When detecting small expression changes, consider more sensitive approaches such as ELISA or mass spectrometry-based techniques to complement antibody-based detection methods.

How can researchers effectively validate ATP2B4 antibody specificity for their particular experimental system?

Comprehensive validation of ATP2B4 antibody specificity requires a multi-faceted approach tailored to each experimental system:

• Genetic manipulation controls: Generate positive and negative controls through overexpression and knockdown/knockout systems. For HAC15 cell models studying ATP2B4 variants, compare antibody signals between wild-type cells, ATP2B4 knockdown cells (using shRNA), and cells expressing ATP2B4 variants to confirm specificity .

• Peptide competition assays: Pre-incubate antibodies with immunizing peptides prior to application in your experimental system. Specific signals should be significantly reduced or eliminated.

• Multi-antibody validation: Test multiple antibodies targeting different ATP2B4 epitopes. Consistent detection patterns across different antibodies increase confidence in specificity.

• Appropriate negative controls: Include tissues/cells known to lack or express minimal ATP2B4, or use isotype control antibodies to assess non-specific binding.

• Mass spectrometry correlation: For the most rigorous validation, perform immunoprecipitation followed by mass spectrometry to confirm the identity of captured proteins.

Document all validation steps in publications, including antibody sources, clone names, catalog numbers, and validation methods employed. This comprehensive approach not only ensures experimental reliability but also provides valuable methodological reference for other researchers working with ATP2B4 antibodies.