ATP1A3 Antibody, Biotin conjugated

What is the target specificity of the ATP1A3 Antibody, Biotin conjugated?

The ATP1A3 antibody with biotin conjugation specifically targets the sodium/potassium-transporting ATPase subunit alpha-3 protein, particularly amino acids 143-278 of human ATP1A3 . This polyclonal antibody, raised in rabbits, recognizes the human ATP1A3 protein, which functions in catalyzing ATP hydrolysis and exchanging sodium and potassium ions across the plasma membrane . The specificity for this particular amino acid region allows researchers to target a distinct epitope of the ATP1A3 protein, making it valuable for detecting this specific region in experimental settings.

What are the storage conditions for maintaining antibody activity?

For optimal preservation of activity, the ATP1A3 antibody with biotin conjugation should be stored at -20°C or -80°C immediately upon receipt . The antibody is stable for approximately one year when properly stored . The formulation includes 50% glycerol with 0.01M PBS at pH 7.4 and 0.03% Proclin 300 as a preservative, which helps maintain stability during storage . Researchers should avoid repeated freeze-thaw cycles as this can compromise antibody performance . For certain size preparations (such as the 20μL size), 0.1% BSA may be included in the formulation to further enhance stability .

What are the validated applications for the biotin-conjugated ATP1A3 antibody?

The primary validated application for the biotin-conjugated ATP1A3 antibody is ELISA (Enzyme-Linked Immunosorbent Assay) . The biotin conjugation specifically enhances detection sensitivity in ELISA applications through strong biotin-streptavidin interactions. While this particular conjugate is optimized for ELISA, other ATP1A3 antibodies with different conjugations or unconjugated forms may be suitable for Western Blot (WB), Immunohistochemistry (IHC), and Immunofluorescence (IF) applications, depending on the specific antibody formulation and the epitope targeted .

How should I design an ELISA protocol using this biotin-conjugated antibody?

When designing an ELISA protocol with the biotin-conjugated ATP1A3 antibody, researchers should follow these methodological steps:

• Coating: Adsorb target antigen or capture antibody to microplate wells

• Blocking: Use appropriate blocking buffer (typically 1-5% BSA in PBS) to reduce non-specific binding

• Primary Antibody: Apply the biotin-conjugated ATP1A3 antibody at recommended dilutions (start with manufacturer's recommendation and optimize)

• Detection System: Use streptavidin-HRP (horseradish peroxidase) conjugate

• Substrate Addition: Add appropriate HRP substrate (TMB or similar)

• Signal Measurement: Measure absorbance at appropriate wavelength

For optimization, perform a titration experiment with different antibody concentrations to determine the optimal signal-to-noise ratio for your specific experimental conditions .

What control samples should be included in experiments using this antibody?

Rigorous experimental design requires several controls when working with the ATP1A3 antibody:

These controls help distinguish true signals from artifacts and validate experimental findings when working with this biotin-conjugated antibody.

How can I minimize background signal when using biotin-conjugated antibodies?

When working with biotin-conjugated antibodies like the ATP1A3 antibody, background signal can arise from endogenous biotin in biological samples. To minimize this interference:

• Implement a biotin-blocking step using streptavidin/avidin before applying the biotin-conjugated antibody

• Use commercial biotin-blocking kits designed specifically for this purpose

• Choose sample preparation methods that minimize biotin release (avoid certain fixatives)

• Optimize antibody concentration through titration experiments

• Extend blocking steps with BSA or specialized blocking reagents

• Include detergents like Tween-20 in washing buffers at appropriate concentrations

These methodological approaches significantly reduce background while preserving specific signal detection of ATP1A3 protein.

What factors might affect the reactivity of the ATP1A3 antibody?

Several factors can influence the reactivity and performance of the ATP1A3 antibody:

• Sample preparation method (fixation type, duration, and protein denaturation conditions)

• Epitope accessibility (the aa 143-278 region may be partially obscured in certain conformations)

• Post-translational modifications of ATP1A3 that may alter epitope recognition

• Buffer composition (pH, salt concentration, detergent type/concentration)

• Incubation conditions (temperature, duration, agitation method)

• Sample storage conditions and age

• Presence of interfering substances in the sample

Systematic optimization of these parameters is essential for achieving optimal results with this antibody across different experimental systems .

How should I address cross-reactivity concerns with this antibody?

While the ATP1A3 antibody (aa 143-278) is designed to be specific for human ATP1A3, researchers should be aware of potential cross-reactivity:

• Perform sequence homology analysis between ATP1A3 and related isoforms (ATP1A1, ATP1A2, ATP1A4) for the 143-278 amino acid region

• Include knockout/knockdown validation experiments when possible

• Run parallel experiments with antibodies targeting different epitopes of ATP1A3

• Consider absorption controls with related proteins

• Analyze samples from species with known sequence divergence in this region

• When interpreting results, consider the presence of homologous proteins, particularly in complex samples

These approaches help establish signal specificity and address legitimate concerns about potential cross-reactivity in research applications .

How can this antibody be used to study ATP1A3's role in mitochondrial stability?

Recent research indicates ATP1A3 may regulate protein synthesis affecting mitochondrial stability under stress conditions . To investigate this function:

• Design co-localization studies combining the biotin-conjugated ATP1A3 antibody with mitochondrial markers

• Develop pull-down assays using the biotin tag to identify protein interaction partners under normal and stress conditions

• Implement proximity ligation assays (PLA) to detect ATP1A3 interactions with mitochondrial proteins

• Utilize the antibody in ChIP-seq or RIP-seq experiments to investigate ATP1A3's potential role in transcriptional or post-transcriptional regulation

• Perform immunoprecipitation followed by mass spectrometry to identify ATP1A3-associated protein complexes in different cellular compartments

These methodological approaches leverage the biotin conjugation and epitope specificity to investigate ATP1A3's emerging role in cellular stress responses and mitochondrial function .

What strategies can be employed to investigate ATP1A3 interactions with RNA-binding proteins?

To examine ATP1A3's reported interactions with RNA-binding proteins , researchers can:

• Use the biotin-conjugated antibody in RNA immunoprecipitation (RIP) assays followed by sequencing or qPCR

• Perform protein-protein interaction studies through pull-down assays utilizing the biotin tag

• Design in vitro binding assays with purified components to assess direct interactions

• Implement FRET or BRET approaches to study these interactions in living cells

• Develop competitive binding assays to identify key interaction domains

• Combine with proximity labeling techniques like BioID or APEX to identify the broader interactome

These advanced applications extend beyond the antibody's basic use in ELISA and leverage the biotin conjugation for sophisticated molecular interaction studies .

How might this antibody be utilized in multiplexed imaging approaches?

The biotin conjugation of this ATP1A3 antibody enables sophisticated multiplexed imaging strategies:

• Combine with streptavidin conjugated to quantum dots for highly stable fluorescence signals

• Implement sequential immunostaining protocols with biotin blocking and stripping steps

• Use in cyclic immunofluorescence (CycIF) protocols where the biotin tag provides consistent detection across cycles

• Combine with tyramide signal amplification for enhanced sensitivity in tissue samples

• Incorporate into imaging mass cytometry workflows for highly multiplexed tissue analysis

• Utilize in expansion microscopy protocols where the biotin-streptavidin interaction withstands the expansion process

These advanced imaging applications extend the utility of this antibody beyond conventional single-target immunofluorescence approaches.

How should discrepancies in molecular weight between predicted and observed ATP1A3 be interpreted?

When analyzing ATP1A3 detection results, researchers may observe discrepancies between the calculated molecular weight (113 kDa) and experimental observations (100-113 kDa) . These variations may result from:

• Post-translational modifications (phosphorylation, glycosylation, ubiquitination)

• Alternative splicing generating different isoforms

• Proteolytic processing in different cellular compartments

• Incomplete protein denaturation affecting gel migration

• Technical variables in SDS-PAGE conditions (buffer composition, acrylamide percentage)

To address these discrepancies, researchers should compare results across multiple techniques (Western blot, mass spectrometry), use multiple antibodies targeting different epitopes, and validate with recombinant protein standards of known molecular weight.

What criteria should be used to validate experimental results with this antibody?

Robust validation of results obtained with the ATP1A3 antibody requires multiple complementary approaches:

• Genetic validation: Compare results between wild-type and ATP1A3 knockout/knockdown samples

• Epitope competition: Pre-incubate antibody with immunizing peptide (aa 143-278) to confirm signal specificity

• Cross-platform validation: Confirm findings using orthogonal techniques (IF, WB, MS)

• Biological replication: Demonstrate reproducibility across independent experiments

• Positive controls: Include tissues/cells known to express ATP1A3 (brain tissue, C2C12 cells)

• Alternative antibodies: Compare results with antibodies targeting different ATP1A3 epitopes

• Functional validation: Correlate protein detection with functional assays of Na+/K+ ATPase activity

How can researchers distinguish between ATP1A3 isoforms and closely related family members?

Distinguishing ATP1A3 from related isoforms (ATP1A1, ATP1A2, ATP1A4) requires careful experimental design:

• Epitope analysis: The aa 143-278 region targeted by this antibody should be compared across isoforms for sequence homology

• Isoform-specific expression patterns: Leverage known tissue distribution differences (ATP1A3 is enriched in neurons)

• Molecular weight differences: ATP1A isoforms have slight MW variations that may be resolved with high-resolution SDS-PAGE

• Co-immunoprecipitation with isoform-specific partners: ATP1A isoforms associate with different beta subunits

• Phosphorylation patterns: Isoform-specific phosphorylation sites can be used for discrimination

• Pharmacological sensitivity: ATP1A isoforms have differential sensitivity to inhibitors like ouabain

These methodological approaches help researchers confidently identify ATP1A3 in complex biological samples and distinguish it from closely related family members .