ATP11B Antibody

What is ATP11B and what cellular functions does it perform?

ATP11B is a widely expressed integral membrane ATPase that functions as a flippase, critical for maintaining the asymmetrical distribution of phosphatidylserine (PS) on the inner leaflet of the cell membrane . It drives the transport of phospholipids across membranes and has been implicated in subnuclear trafficking of transcription factors with RING motifs . Recent research has identified ATP11B as a potent metastatic suppressor, particularly in breast cancer, where low expression of ATP11B coupled with high PTDSS2 expression is associated with enhanced metastasis through increased nonapoptotic phosphatidylserine exposure on the outer cell membrane leaflet .

What applications is the ATP11B antibody suitable for in laboratory research?

The ATP11B antibody has been validated for multiple research applications including:

Researchers should note that the antibody reacts with both human and mouse ATP11B, making it versatile for cross-species studies .

How should ATP11B antibody be stored to maintain its efficacy?

For optimal performance, ATP11B antibody should be stored at 4°C for short-term use (up to three months) or at -20°C for long-term storage (up to one year) . Repeated freeze-thaw cycles should be avoided as they can compromise antibody integrity and binding efficiency. The antibody is typically supplied in PBS containing 0.02% sodium azide, which helps maintain stability . For researchers planning extended studies, aliquoting the antibody upon receipt is recommended to minimize freeze-thaw cycles.

What controls should be included when using ATP11B antibody in Western blot experiments?

When designing Western blot experiments with ATP11B antibody, researchers should include:

• Positive control: K562 cell lysate has been validated as an appropriate positive control .

• Negative control: Include samples where ATP11B is not expressed or is knocked down.

• Peptide competition assay: Run parallel samples with and without blocking peptide to confirm specificity, as shown in validation studies where the 68 kDa band was eliminated in the presence of blocking peptide .

• Loading control: Include appropriate housekeeping proteins (β-actin, GAPDH, etc.) to normalize protein loading.

• Molecular weight markers: ATP11B has an observed molecular weight of 68 kDa, which differs from its calculated weight of 134 kDa, making proper markers essential for accurate identification .

This comprehensive approach ensures reliable identification of ATP11B and minimizes false positive/negative results.

What are the optimal sample preparation methods for detecting ATP11B in different cellular compartments?

ATP11B is primarily localized in cell membranes as a phospholipid transporter. For optimal detection:

• For whole cell lysates: Use RIPA buffer supplemented with protease inhibitors, keeping samples cold throughout processing.

• For membrane fraction enrichment: Employ differential centrifugation protocols to isolate membrane fractions.

• For immunofluorescence applications:

  • Fix cells with 4% paraformaldehyde for 10-15 minutes

  • Permeabilize with 0.1-0.2% Triton X-100

  • Block with 3-5% BSA or normal serum

  • Incubate with primary antibody at 20 μg/mL overnight at 4°C

  • Use appropriate fluorophore-conjugated secondary antibodies

Subcellular fractionation is recommended when studying ATP11B's distribution between plasma membrane and intracellular compartments, as its localization can provide insights into its functional status in experimental models .

How can researchers differentiate between ATP11B and other P-type ATPases in experimental systems?

Distinguishing ATP11B from other P-type ATPases requires:

• Antibody specificity: The ATP11B antibody used should be raised against unique epitopes. The antibody described in the search results targets a 19-amino acid synthetic peptide near the amino terminus of human ATP11B, located within amino acids 290-340 .

• Molecular weight verification: ATP11B has an observed molecular weight of 68 kDa in Western blot applications, which can help differentiate it from other similar ATPases .

• Genetic approaches: For definitive differentiation, consider using:

  • ATP11B-specific siRNA/shRNA knockdown as controls

  • CRISPR/Cas9-mediated knockout of ATP11B, which has been successfully used in research to validate its function

  • Selective expression of catalytically inactive mutants (e.g., E186K in mouse or E180K in human) that disrupt the DGET motif essential for ATP11B function

• Functional assays: Measure phospholipid flippase activity with fluorescently-labeled phospholipid analogs, as ATP11B specifically maintains PS on the inner leaflet of the membrane.

How can ATP11B antibody be used to investigate its role in cancer metastasis pathways?

Based on recent discoveries of ATP11B as a metastatic suppressor in breast cancer, researchers can use ATP11B antibody to:

• Profile ATP11B expression across tumor samples using immunohistochemistry or Western blotting to correlate expression levels with metastatic potential.

• Combine with PTDSS2 antibodies for dual staining, as the ATP11B^lo PTDSS2^hi phenotype has been identified as a marker for enhanced metastasis .

• Assess ATP11B expression changes in response to therapeutic interventions, particularly in combination therapy models involving anti-PS antibodies with paclitaxel or docetaxel .

• Investigate cellular responses to ATP11B manipulation by quantifying:

  • Phosphatidylserine exposure using fluorescently labeled annexin V

  • Changes in immune cell infiltration, particularly myeloid-derived suppressor cells

  • Alterations in cytotoxic T cell activity in the tumor microenvironment

• Conduct co-immunoprecipitation studies to identify ATP11B-interacting proteins involved in metastatic pathways.

The research indicates that ATP11B expression status could serve as a potential biomarker for metastatic risk and therapeutic response in breast cancer patients .

What approaches can be used to study ATP11B catalytic activity in relation to phosphatidylserine distribution?

To investigate ATP11B's flippase activity and its impact on phosphatidylserine distribution:

• Phospholipid flippase assays:

  • Use fluorescently labeled PS analogs to track movement across the membrane

  • Compare PS distribution in ATP11B wildtype versus ATP11B-depleted or mutant cells

• Flow cytometry with annexin V:

  • Measure PS exposure on the outer leaflet of cell membranes

  • Distinguish apoptotic from non-apoptotic PS exposure using appropriate viability dyes

• Genetic manipulation strategies:

  • Express catalytically inactive mutants (E186K in mouse, equivalent to human E180K) to study the functional consequences of disrupted ATP11B activity

  • Use CRISPR/Cas9-mediated knockout to completely eliminate ATP11B expression

• Domain-specific analysis:

  • Study the four main domains of ATP11B (cytoplasmic A, N, P, and R domains)

  • Focus on the DGET motif (amino acids 178-181 in humans) in the A domain that is critical for dephosphorylation of the phosphorylated intermediate

Research has demonstrated that cells with catalytically inactive ATP11B (E186K mutation) phenocopy ATP11B-depleted cells regarding PS displacement and metastatic potential, confirming the importance of ATP11B's enzymatic activity in its tumor suppressive function .

How can researchers investigate the relationship between ATP11B expression and immune cell responses in the tumor microenvironment?

To explore how ATP11B affects immune responses in the tumor microenvironment:

• Multiplex immunostaining approaches:

  • Use ATP11B antibody in combination with immune cell markers

  • Quantify spatial relationships between ATP11B-expressing tumor cells and infiltrating immune populations

• Flow cytometry and CyTOF analysis:

  • Assess myeloid-derived suppressor cell (MDSC) populations in relation to ATP11B status

  • Research has shown significant increases in both monocytic MDSCs (CD11b+Ly6C+) and PMN-MDSCs (CD11b+Ly6G+) in ATP11B-depleted tumors

• Functional immune assays:

  • T cell cytotoxicity assays against ATP11B-normal versus ATP11B-depleted cancer cells

  • Cytokine profiling in the tumor microenvironment related to ATP11B status

• In vivo models:

  • Implant control or sgATP11B-expressing cancer cells into immunocompetent and immunodeficient mice

  • Compare metastatic patterns and immune infiltrates between models

• Therapeutic intervention studies:

  • Test anti-PS antibodies in combination with chemotherapeutics in ATP11B-low tumors

  • Monitor changes in immune cell composition and activation status following treatment

The data indicates that the immunosuppressive environment created by ATP11B depletion contributes significantly to enhanced metastasis, suggesting potential immunotherapeutic approaches for patients with low ATP11B expression .

What are the most common technical issues when using ATP11B antibody and how can they be resolved?

When working with ATP11B antibody, researchers might encounter several challenges:

• High background in immunostaining:

  • Increase blocking time or concentration (5% BSA or normal serum)

  • Optimize antibody concentration (start with recommended 10-20 μg/mL for ICC/IF and adjust as needed)

  • Include 0.1% Tween-20 in wash buffers

  • Ensure secondary antibody compatibility and specificity

• Weak or no signal in Western blots:

  • Ensure sufficient protein loading (50-100 μg total protein)

  • Optimize transfer conditions for large proteins (wet transfer recommended)

  • Increase antibody concentration or incubation time

  • Use enhanced chemiluminescence substrate for detection

• Multiple bands in Western blot:

  • Note that ATP11B has a calculated molecular weight of 134 kDa but is observed at 68 kDa in some systems

  • Use peptide competition controls to identify specific binding

  • Consider post-translational modifications or isoforms

• Inconsistent results between experiments:

  • Maintain consistent sample preparation protocols

  • Prepare fresh working solutions of antibody

  • Store antibody properly to prevent degradation (avoid freeze-thaw cycles)

• Cross-reactivity concerns:

  • The antibody is documented to react with human and mouse ATP11B

  • For other species, validation experiments are required

How should researchers quantify and normalize ATP11B expression in comparative studies?

For reliable quantification of ATP11B expression:

• Western blot densitometry:

  • Use housekeeping proteins (β-actin, GAPDH, tubulin) for normalization

  • Include a standard curve with known amounts of recombinant protein when absolute quantification is needed

  • Analyze using appropriate software (ImageJ, Image Lab, etc.)

  • Report results as fold-change relative to control samples

• Immunohistochemistry/immunofluorescence quantification:

  • Use consistent exposure settings and acquisition parameters

  • Quantify signal intensity using appropriate software

  • Normalize to cell number or tissue area

  • Consider implementing H-score or Allred scoring systems for consistency

• qPCR for transcript analysis:

  • Use validated reference genes for normalization

  • Implement the 2^-ΔΔCt method for relative quantification

  • Consider absolute quantification using standard curves when comparing across different experimental systems

• Statistical considerations:

  • Perform at least three independent biological replicates

  • Apply appropriate statistical tests based on data distribution

  • Set significance thresholds a priori

Research indicates significant variability in ATP11B expression between normal and tumor tissues, with expression in BRCA1-mutant primary tumors approximately 10% of wild-type levels, necessitating careful quantification approaches .

How can ATP11B antibody be utilized in developing new therapeutic approaches for metastatic cancer?

The discovery of ATP11B's role as a metastatic suppressor opens several avenues for therapeutic development:

• Biomarker development:

  • Use ATP11B antibody to screen patient samples for ATP11B expression levels

  • Develop immunohistochemistry-based diagnostic tests to identify patients with ATP11B^lo PTDSS2^hi phenotype who might benefit from targeted therapies

• Therapeutic target identification:

  • Screen for compounds that can restore or enhance ATP11B expression or activity

  • Investigate combination therapies that target both ATP11B-low status and its downstream effects

• Personalized medicine approaches:

  • Stratify patients based on ATP11B/PTDSS2 expression for clinical trials

  • Monitor ATP11B expression changes during treatment response and resistance development

• Combinatorial therapy development:

  • Expand on findings that anti-PS antibodies with paclitaxel or docetaxel can effectively overcome metastatic processes in ATP11B^lo PTDSS2^hi cancer cells

  • Investigate immunotherapy combinations targeting the immunosuppressive environment associated with low ATP11B expression

• Drug resistance research:

  • Investigate whether ATP11B expression changes correlate with resistance to anti-cancer drugs, similar to the observed increased expression of the homologous ATP11A in cells resistant to farnesyltransferase inhibitors

What are the emerging techniques for studying ATP11B localization and dynamics in living cells?

Advanced methodologies for investigating ATP11B dynamics include:

• Live-cell imaging approaches:

  • Generate fluorescently tagged ATP11B constructs (GFP, mCherry fusions)

  • Implement FRAP (Fluorescence Recovery After Photobleaching) to study ATP11B mobility

  • Use FRET-based biosensors to monitor ATP11B interactions with other proteins or lipids

• Super-resolution microscopy:

  • Apply STORM, PALM, or STED microscopy to visualize ATP11B distribution at nanoscale resolution

  • Combine with fluorescently labeled phospholipids to track ATP11B-mediated lipid movement

• Optogenetic approaches:

  • Develop light-controlled ATP11B variants to manipulate its activity in specific cellular compartments

  • Study the temporal aspects of ATP11B function in phospholipid translocation

• Mass spectrometry imaging:

  • Map phospholipid distribution in relation to ATP11B localization

  • Quantify changes in membrane composition following ATP11B manipulation

• Cryo-electron microscopy:

  • Determine the structural details of ATP11B alone and in complex with transported lipids

  • Investigate structural changes associated with catalytically inactive mutants like E186K

These advanced techniques will help elucidate the dynamic behavior of ATP11B in cellular membranes and provide deeper insights into its mechanism of action in both normal and pathological conditions.

What are the most significant unresolved questions regarding ATP11B function and regulation?

Despite recent advances, several critical questions about ATP11B remain unanswered:

• Substrate specificity: While ATP11B is known to transport phospholipids, the exact molecule(s) it transports and its selectivity among different phospholipids requires further clarification .

• Regulatory mechanisms: How ATP11B expression and activity are regulated under normal physiological conditions and in disease states remains poorly understood.

• Interaction network: The complete protein-protein interaction network for ATP11B, including potential regulatory partners and downstream effectors, needs comprehensive characterization.

• Tissue-specific functions: Whether ATP11B performs specialized functions in different tissues and cell types beyond its general role in phospholipid transport requires investigation.

• Therapeutic targeting: The feasibility of directly targeting ATP11B for therapeutic purposes, particularly in cancer, needs exploration through high-throughput screening approaches.

• Relationship with BRCA1: The mechanistic details of how BRCA1 regulates ATP11B expression and the implications for hereditary breast cancer require deeper investigation .

• Role in drug resistance: The potential involvement of ATP11B in resistance to cancer therapeutics, suggested by the observed relationship between its homolog ATP11A and resistance to farnesyltransferase inhibitors, warrants systematic study .