ATP2B2 Antibody

What is ATP2B2 and why is it important in research?

ATP2B2 (ATPase Plasma Membrane Ca²⁺ Transporting 2), also known as PMCA2, is a plasma membrane calcium ATPase that plays a critical role in maintaining calcium homeostasis in cells. It functions by removing calcium ions from cells against large concentration gradients.

ATP2B2 is particularly important in research because:

• It is predominantly expressed in the brain cortex

• It plays a crucial role in calcium homeostasis in auditory hair cells

• Mutations in ATP2B2 are associated with hearing loss and ataxia

• It has been identified as a potential antibody-drug conjugate target in cancer research

The protein has a molecular weight of approximately 137 kDa and up to 8 different isoforms have been reported . Its proper function is essential for neuronal signaling, auditory transduction, and various cellular processes dependent on calcium regulation.

What are the common applications for ATP2B2 antibodies?

ATP2B2 antibodies are utilized across multiple experimental applications:

When selecting an application, consider that membrane proteins like ATP2B2 may require specific sample preparation protocols to maintain native conformation .

How should ATP2B2 antibodies be stored and handled?

Proper storage and handling of ATP2B2 antibodies are crucial for maintaining their effectiveness:

• Storage temperature: Most ATP2B2 antibodies should be stored at -20°C

• Long-term stability: Valid for approximately 12 months when properly stored

• Avoid freeze/thaw cycles: Repeated freezing and thawing can degrade antibody quality

• Buffer solution: Typically provided in phosphate buffered solution (pH 7.4) containing stabilizers and glycerol

• Shipping: Usually shipped with ice packs and should be stored immediately upon receipt

For optimal results, aliquot antibodies upon first thaw to minimize freeze/thaw cycles, and follow manufacturer-specific recommendations for each antibody clone.

How do I choose the most appropriate ATP2B2 antibody for my experiment?

Selecting the optimal ATP2B2 antibody requires consideration of several factors:

• Target specificity: Determine which region of ATP2B2 you want to target (N-terminal, C-terminal, specific domains)

• Species reactivity: Verify the antibody reacts with your species of interest. Common reactivities include:

  • Human and rat

  • Mouse, rat

  • Predicted reactivity for bovine, horse, sheep, rabbit, dog

• Application compatibility: Ensure the antibody is validated for your specific application

• Clonality considerations:

  • Polyclonal: Better for detecting denatured proteins, potentially higher sensitivity but lower specificity

  • Monoclonal: Higher specificity but may be less robust to protein modifications

• Validation status: Check literature citations and validation data to ensure reliability

Compare data from resources like Antibodypedia, which lists top validated ATP2B2 antibodies with reference counts .

What controls should I include when using ATP2B2 antibodies?

Proper controls are essential for reliable interpretation of results with ATP2B2 antibodies:

• Positive controls:

  • Brain cortex tissue (highest expression)

  • Cell lines known to express ATP2B2

• Negative controls:

  • Primary antibody omission

  • Tissues/cells with confirmed absence of ATP2B2

  • Isotype control (same concentration as primary antibody)

• Loading/technical controls:

  • For Western blot: β-actin (as demonstrated in studies)

  • For IHC/IF: DAPI for nuclear counterstaining

• Validation controls:

  • Peptide competition assay using the immunizing peptide

  • Knockdown/knockout samples if available

These controls help distinguish specific from non-specific binding and validate experimental techniques.

How can I optimize Western blot protocols for ATP2B2 detection?

Optimizing Western blot for ATP2B2 requires attention to several critical parameters:

• Sample preparation:

  • For membrane proteins like ATP2B2, use appropriate lysis buffers containing detergents (e.g., 0.5% Triton X-100, 0.1% SDS)

  • Include protease inhibitors to prevent degradation

  • For brain tissue samples, flash freezing and proper homogenization are critical

• Gel selection and transfer:

  • Use 4-20% gradient gels to effectively resolve the 137 kDa ATP2B2 protein

  • Consider longer transfer times for this large protein

• Antibody optimization:

  • Typical dilutions range from 1:1000-3000

  • Block with 5% non-fat milk or BSA in TBS-T

  • Consider overnight primary antibody incubation at 4°C

• Detection optimization:

  • ECL Plus detection reagent has been successfully used in published protocols

  • Longer exposure times may be needed for lower expression samples

• Quantification approach:

  • Normalize to loading controls like β-actin

  • Use non-manipulated images for quantification via software like ImageJ

Following these guidelines will help achieve clear and specific detection of ATP2B2 protein in Western blot applications.

How can ATP2B2 antibodies be used to investigate hearing loss mechanisms?

ATP2B2 plays a crucial role in auditory function, and antibodies against this protein have been instrumental in studying hearing loss mechanisms:

• Localization studies:

  • ATP2B2 antibodies reveal the protein's distribution in stereocilia bundles of auditory hair cells

  • Immunohistochemistry with ATP2B2 antibodies can detect alterations in protein localization in disease models

• Expression analysis:

  • Western blot quantification can determine if ATP2B2 protein levels are altered in hearing loss models

  • Immunofluorescence intensity measurements can detect regional changes in expression

• Functional correlation:

  • Studies using ATP2B2 antibodies have shown how mutations affect calcium homeostasis in stereocilia

  • ATP2B2 antibodies help visualize the relationship between protein localization and functional calcium clearance

• Genetic interaction studies:

  • ATP2B2 antibodies have helped establish how this protein interacts with other hearing-related proteins, such as cadherin 23 (CDH23)

  • Researchers have used these antibodies to demonstrate how the Atp2b2 deafwaddler allele (dfwi5) affects protein expression and function

These approaches have been critical in establishing ATP2B2's role in calcium clearance from stereocilia and maintaining appropriate calcium levels in the endolymph, both essential for normal auditory function.

What considerations are important when using ATP2B2 antibodies in immunohistochemistry of neuronal tissues?

Neuronal tissue presents unique challenges for ATP2B2 immunohistochemistry:

Studies have shown that ATP2B2 is mainly expressed in brain cortex , making these considerations particularly important for accurately detecting physiological expression patterns.

How can ATP2B2 antibodies be used to study calcium signaling dysregulation in disease models?

ATP2B2 antibodies provide valuable tools for investigating calcium dysregulation in various disease contexts:

• Quantitative analysis approaches:

  • Western blotting with ATP2B2 antibodies can quantify expression changes in disease models

  • Immunofluorescence intensity analysis can measure regional expression differences

• Subcellular localization studies:

  • High-resolution confocal microscopy with ATP2B2 antibodies can reveal altered subcellular distribution

  • Co-localization with organelle markers can identify trafficking defects

• Dynamic regulation assessment:

  • ATP2B2 antibodies can be used to track protein expression changes after calcium challenge

  • Time-course immunocytochemistry can reveal adaptive responses to calcium dysregulation

• Disease-specific applications:

  • In hereditary hearing loss: ATP2B2 antibodies help visualize stereocilia defects

  • In cerebellar ataxia: Immunohistochemistry can reveal altered expression in Purkinje cells

  • In cancer research: ATP2B2 antibodies help evaluate its potential as an antibody-drug conjugate target

Research has demonstrated that functional characterization using calcium export assays coupled with ATP2B2 antibody detection can reveal how specific variants (e.g., c.3028G>A, p.(Glu1010Lys)) compromise calcium export capacity compared to wild type .

Why might I observe multiple bands when using ATP2B2 antibodies in Western blot?

Multiple bands in ATP2B2 Western blots can occur for several reasons:

• Alternative splicing:

  • ATP2B2 has up to 8 different isoforms reported , which can appear as distinct bands

  • Tissue-specific expression of variants may lead to different banding patterns

• Post-translational modifications:

  • Phosphorylation or glycosylation can alter protein migration

  • Different states of modification may appear as multiple bands

• Protein degradation:

  • Partial proteolysis during sample preparation can generate fragments

  • Include fresh protease inhibitors in lysis buffer to minimize this issue

• Cross-reactivity:

  • Antibodies may detect related calcium ATPases (ATP2B1, ATP2B3, ATP2B4)

  • Use purified antibodies with validated specificity for ATP2B2

• Experimental artifacts:

  • Incomplete denaturation can cause aggregates or alternative conformations

  • Ensure complete sample denaturation with sufficient SDS and heating

As noted by Elabscience, "The observed MW is not always consistent with the expectation. The mobility is affected by many factors, which may cause the observed band size to be inconsistent with the expected size."

How can I minimize background when using ATP2B2 antibodies in immunofluorescence?

High background in ATP2B2 immunofluorescence can compromise results. Here are strategies to minimize it:

• Antibody optimization:

  • Titrate antibody concentration (recommended ranges: 1:100-1:500 for IF/ICC)

  • Use affinity-purified antibodies when available

• Blocking optimization:

  • Extend blocking time (1-2 hours at room temperature)

  • Use alternative blocking agents (BSA, fish gelatin, or commercial blockers)

  • Include 0.1-0.3% Triton X-100 in blocking solution for better penetration

• Washing optimization:

  • Increase washing duration and number of washes

  • Add 0.1% Tween-20 to wash buffer to reduce non-specific binding

• Fixation considerations:

  • Overfixation can increase background; optimize fixation time

  • Consider alternative fixatives (methanol, acetone) for specific antibodies

• Controls to identify sources of background:

  • Secondary antibody only control identifies non-specific secondary binding

  • Isotype control identifies Fc receptor binding

• Signal-to-noise enhancement:

  • Use low autofluorescence mounting media

  • Consider a Sudan Black B treatment to reduce tissue autofluorescence

These approaches help ensure that observed signals accurately represent ATP2B2 expression rather than artifacts.

How do I distinguish between specific and non-specific binding in ATP2B2 immunohistochemistry?

Distinguishing specific from non-specific binding is critical for accurate interpretation of ATP2B2 immunohistochemistry:

• Critical controls:

  • Primary antibody omission: Should show no signal

  • Isotype control: Uses irrelevant antibody of same isotype and concentration

  • Peptide competition: Pre-incubation with immunizing peptide should abolish specific signal

  • Genetic knockdown/knockout tissue (when available): Should show reduced/absent signal

• Pattern assessment:

  • Compare observed staining with known expression pattern (primarily brain cortex for ATP2B2)

  • Cell membrane localization is expected for ATP2B2

  • Non-specific staining often appears as diffuse background or nuclear staining

• Validation approaches:

  • Use two different antibodies targeting distinct epitopes of ATP2B2

  • Compare protein localization with known mRNA expression data

  • Correlate with functional data

• Technical considerations:

  • Optimize antigen retrieval to enhance specific binding

  • Adjust primary antibody concentration to improve signal-to-noise ratio

  • Consider signal amplification methods for low-expression samples

Studies have consistently shown ATP2B2 to be primarily localized to the plasma membrane, particularly in stereocilia of hair cells in the inner ear and in specific neuronal populations .

How are ATP2B2 antibodies being used in cancer research and antibody-drug conjugate development?

ATP2B2 has emerged as a potential target for antibody-drug conjugate (ADC) development in cancer therapy:

• Target validation approaches:

  • ATP2B2 antibodies help confirm protein expression in tumor samples versus normal tissues

  • Immunohistochemistry using ATP2B2 antibodies has identified this protein as having low or undetectable expression across 44 normal tissues

• Expression profiling:

  • ATP2B2 was identified among 82 prioritized targets for ADC development through comprehensive screening

  • ATP2B2 showed low/not detected expression across all 13 critical normal tissues, suggesting potential therapeutic window

• Multi-omics integration:

  • Research combined transcriptomics, proteomics, immunohistochemistry and cell surface membrane datasets to identify ATP2B2 as a potential ADC target

  • ATP2B2 antibodies were crucial for validating expression patterns identified in transcriptomic analyses

• Therapeutic development applications:

  • ATP2B2 antibodies help assess potential on-target/off-tumor effects

  • ATP2B2 was identified among 15 targets (including AQP5, CSPG5, EDNRB, MSLN, MUC16) exhibiting low/not detected expression in critical normal tissues

These findings suggest ATP2B2 may have potential as an ADC target with favorable tumor-to-normal tissue expression ratios, particularly for specific cancer types.

What role do ATP2B2 antibodies play in studying genetic hearing loss mechanisms?

ATP2B2 antibodies have been instrumental in elucidating hearing loss mechanisms:

• Mutation phenotyping:

  • ATP2B2 antibodies help characterize how specific mutations affect protein expression, localization, and function

  • Studies using ATP2B2 antibodies revealed that loss of auditory function in PMCA2 mutants can be attributed to dysregulation of intracellular Ca²⁺ inside stereocilia bundles

• Protein-protein interaction studies:

  • Immunoprecipitation with ATP2B2 antibodies helps identify binding partners

  • Research has demonstrated interaction between ATP2B2 and CDH23 (cadherin 23), a component of stereocilia tip-links

• Therapeutic development evaluation:

  • ATP2B2 antibodies help assess outcomes of therapeutic interventions

  • In a recent study, CRISPR-Cas9 treatment targeting the Atp2b2 Oblivion mutation was evaluated using antibodies to confirm protein expression changes

• Gene variant characterization:

  • Antibodies help determine if variants affect protein stability

  • Western blotting with ATP2B2 antibodies helped characterize the novel null Atp2b2 allele, dfwi5

This research has significant implications, as "liposome-mediated in vivo delivery of CRISPR-Cas9 ribonucleoprotein complexes leads to specific editing of the Atp2b2 Oblivion allele" resulting in "outer hair cell survival and restores their function, leading to hearing recovery" .

How can I use ATP2B2 antibodies to investigate neurological disorders associated with calcium dysregulation?

ATP2B2 antibodies provide valuable tools for investigating calcium-related neurological disorders:

• Expression analysis in pathological samples:

  • Quantitative immunohistochemistry can reveal altered ATP2B2 expression in affected brain regions

  • Western blot analysis can quantify protein level changes in disease versus control tissues

• Functional correlation approaches:

  • Combine ATP2B2 antibody labeling with calcium imaging to correlate protein expression with functional calcium handling

  • Co-labeling with markers of neuronal stress or death helps establish causal relationships

• Genetic variant characterization:

  • ATP2B2 antibodies can assess how specific variants affect protein expression and localization

  • This approach helped characterize the c.3028G>A, p.(Glu1010Lys) variant in a case of recurrent post-infectious cerebellar ataxia

• Therapeutic response assessment:

  • ATP2B2 antibodies can monitor protein expression changes following treatment

  • In a case study, immunotherapy with IVIg was effective in a patient with an ATP2B2 variant, suggesting "immunotherapy with IVIg may augment clinical outcomes in those with pathogenic ATP2B2 gene variants"