ATP2B4 Antibody, FITC conjugated

What is ATP2B4 and what role does it play in cellular calcium homeostasis?

ATP2B4, also known as PMCA4 (Plasma Membrane Ca²⁺-ATPase 4), is a critical calcium transporter in the plasma membrane with a molecular weight of approximately 129,403 Da . This protein functions as an essential regulator of intracellular calcium concentration by actively pumping calcium ions out of cells against concentration gradients. The protein is encoded by the ATP2B4 gene, which has several aliases including MXRA1, ATP2B2, PMCA4b, PMCA4, and PMCA4x . ATP2B4/PMCA4 is particularly important in non-excitable cells and plays a crucial role in maintaining calcium homeostasis across the plasma membrane of various cell types, including red blood cells, where dysregulation can lead to pathological conditions .

What applications are validated for ATP2B4 Antibody, FITC conjugated?

ATP2B4 Antibody, FITC conjugated has been validated for several research applications. According to product information, the antibody has been validated for EIA (Enzyme Immunoassay), general Immunoassay procedures, and ELISA (Enzyme-Linked Immunosorbent Assay) . While the antibody's FITC conjugation makes it particularly suitable for flow cytometry and fluorescence microscopy applications, researchers should verify specific application suitability with the manufacturer before designing experiments . For immunofluorescence applications, available studies have used ATP2B4 antibodies for indirect cellular immunofluorescence labeling of the surface of red blood cells, which can be visualized using confocal microscopy at appropriate excitation wavelengths .

What are the technical specifications of commercially available ATP2B4 Antibody, FITC conjugated?

ATP2B4 Antibody, FITC conjugated is typically available as a rabbit polyclonal antibody with IgG isotype . These antibodies recognize human ATP2B4 antigen and are supplied in liquid format with high purity (>95%), having been purified using Protein G . The antibodies target specific amino acid regions of the ATP2B4 protein, such as amino acids 281-345, as seen in certain products . The antibody's conjugation to FITC (Fluorescein Isothiocyanate) enables direct fluorescent visualization without the need for secondary antibodies in appropriate applications . Most commercial preparations are validated for human reactivity, and researchers should carefully check cross-reactivity with other species if working with non-human models .

How should ATP2B4 Antibody, FITC conjugated be stored and handled to maintain optimal activity?

While specific storage conditions may vary slightly by manufacturer, ATP2B4 Antibody, FITC conjugated should generally be stored at 2-8°C for short-term use (up to 1 month) and at -20°C for longer periods . As a FITC-conjugated product, the antibody is light-sensitive, so storage in dark conditions is essential to prevent photobleaching of the fluorophore. Working dilutions should be prepared fresh before use, and repeated freeze-thaw cycles should be avoided to maintain antibody integrity. When handling the antibody, use sterile techniques and appropriate personal protective equipment. Before use in experiments, allow the antibody to reach room temperature and gently mix (do not vortex) to ensure homogeneity without damaging the protein structure.

How can ATP2B4 Antibody, FITC conjugated be used to investigate calcium dysregulation in multiple myeloma (MM)?

Multiple myeloma-associated hypercalcemia affects various cellular functions, including those in red blood cells. Researchers can use ATP2B4 Antibody, FITC conjugated to investigate the expression and localization of PMCA4 in MM patient samples compared to healthy controls. A methodological approach based on current research includes:

• Collect red blood cells from MM patients and healthy controls

• Prepare RBC smears and fix with methanol

• Permeabilize with saponin (e.g., P0095, Beyotime) and block with appropriate blocking buffer

• Label RBC membrane with DiD (D4019, excitation at 633 nm)

• Apply ATP2B4 Antibody, FITC conjugated for direct detection or use unconjugated primary ATP2B4 antibody followed by FITC-conjugated secondary antibody

• Image using confocal microscopy with appropriate excitation wavelengths (405 nm for certain secondary antibodies, 488 nm for FITC)

• Quantify fluorescence intensity using ImageJ software

Research has shown that PMCA4 expression is significantly reduced in MM red blood cells compared to normal controls, correlating with calcium dysregulation . This approach allows researchers to visualize both the distribution and relative quantity of PMCA4 on cell membranes.

What protocols have been optimized for immunofluorescence studies of ATP2B4 in red blood cells?

For immunofluorescence studies of ATP2B4 in red blood cells, researchers have optimized the following protocol based on published research:

• Fix RBC smears with methanol

• Permeabilize cell membranes with saponin (P0095, Beyotime)

• Block nonspecific binding with QuickBlockTM blocking buffer (P0260, Beyotime)

• Fluorescently label the erythrocyte membrane with 10 μM DiD working solution (D4019)

• Apply ATP2B4/PMCA4 antibody at a dilution ratio of 1:400

• Apply secondary antibody such as anti-mouse IgG (H+L) CFTM 405S at 1:100 dilution

• Observe fluorescence using confocal laser microscopy at appropriate excitation wavelengths (405 nm for blue fluorescence and 633 nm for red fluorescence)

• Analyze fluorescence intensity quantitatively using ImageJ software

This protocol allows for clear visualization of PMCA4 distribution on the RBC membrane and comparative analysis between different sample groups. The dual labeling approach enables researchers to confirm membrane localization of the protein while assessing relative expression levels.

How does miR-4261 regulate ATP2B4 expression, and how can this be studied using ATP2B4 antibodies?

Research indicates that miR-4261 can target and regulate the ATP2B4 gene, reducing PMCA4 protein expression . To study this regulatory relationship, researchers can use ATP2B4 antibodies in combination with molecular techniques:

• First, confirm the targeting relationship using dual-luciferase reporter assays with wild-type and mutant 3'-UTR fragments of ATP2B4 mRNA

• Transfect cells with miR-4261 mimics or negative control

• At different time points post-transfection (36, 48, and 72 hours), collect cells for:

  • qRT-PCR analysis of ATP2B4 mRNA levels using appropriate primers

  • Western blotting to assess PMCA4 protein levels using ATP2B4 antibodies

  • Immunofluorescence analysis using ATP2B4 Antibody, FITC conjugated to visualize changes in protein localization and expression

Studies have shown that miR-4261 significantly reduces both mRNA and protein levels of ATP2B4, with the most pronounced effect at 72 hours post-transfection . Using FITC-conjugated ATP2B4 antibodies in flow cytometry or confocal microscopy enables researchers to quantify these changes at the single-cell level and assess potential heterogeneity in response.

What considerations should be taken when optimizing signal-to-noise ratio in ATP2B4 Antibody, FITC conjugated experiments?

Optimizing signal-to-noise ratio is critical when using ATP2B4 Antibody, FITC conjugated, especially in samples with potentially low expression levels. Consider these methodological approaches:

• Fixation optimization: Test different fixation methods (paraformaldehyde vs. methanol) to determine which best preserves ATP2B4 epitope accessibility while maintaining membrane integrity

• Permeabilization calibration: If intracellular epitopes are targeted, titrate permeabilization agents (saponin concentration and exposure time) to allow antibody access while minimizing nonspecific binding

• Blocking protocol: Use appropriate blocking buffers (QuickBlockTM has been validated ) and optimize blocking time to reduce background

• Antibody titration: Perform dilution series of ATP2B4 Antibody, FITC conjugated to determine optimal concentration that maximizes specific signal while minimizing background

• Counterstaining strategy: When using membrane dyes like DiD , ensure spectral separation from FITC to avoid bleed-through

• Microscopy settings: Adjust laser power, gain, and offset settings on confocal microscopes to capture full dynamic range of fluorescence while avoiding pixel saturation

• Controls inclusion: Always include negative controls (isotype controls, secondary-only controls) and positive controls (samples known to express ATP2B4) in each experiment

Researchers studying ATP2B4 in red blood cells have successfully used these approaches to visualize and quantify significant differences in PMCA4 expression between multiple myeloma samples and normal controls .

How can ATP2B4 Antibody, FITC conjugated be used alongside functional calcium assays to correlate protein expression with calcium transport activity?

Integrating ATP2B4 Antibody, FITC conjugated detection with functional calcium assays provides powerful insights into the relationship between PMCA4 expression and calcium transport activity. A comprehensive methodological approach includes:

• Flow cytometry with calcium indicators:

  • Stain cells with ATP2B4 Antibody, FITC conjugated

  • Co-stain with calcium indicators (Fluo-4, Fura-2)

  • Perform flow cytometry to correlate ATP2B4 expression with basal calcium levels at single-cell resolution

• Live cell imaging with calcium perturbation:

  • In cells transfected with fluorescently-tagged ATP2B4 variants, perform calcium imaging

  • Apply calcium ionophores or physiological stimuli to induce calcium flux

  • Measure calcium recovery rates in cells with different ATP2B4 expression levels

• Atomic absorption spectroscopy correlation:

  • Sort cells based on ATP2B4 Antibody, FITC conjugated staining intensity

  • Perform atomic absorption spectroscopy to quantify absolute calcium content in different populations

  • Correlate calcium content with ATP2B4 expression levels

• Pharmacological intervention:

  • Use specific PMCA inhibitors (like Omega-Agatoxin IVA as used in related research )

  • Compare calcium dynamics between inhibited and non-inhibited samples

  • Correlate findings with ATP2B4 antibody staining patterns

This multimodal approach enables researchers to establish not just correlative but potentially causative relationships between ATP2B4/PMCA4 expression levels and cellular calcium handling capacity, particularly in pathological conditions like multiple myeloma where calcium dysregulation is implicated .

What are common technical challenges when using ATP2B4 Antibody, FITC conjugated, and how can they be addressed?

Researchers working with ATP2B4 Antibody, FITC conjugated may encounter several technical challenges:

• Low signal intensity:

  • Cause: Insufficient antibody concentration, poor epitope accessibility, or low target expression

  • Solution: Optimize antibody concentration through titration; try alternative fixation methods; amplify signal using additional layers of detection (e.g., anti-FITC antibodies) where appropriate

• High background fluorescence:

  • Cause: Nonspecific binding, inadequate blocking, autofluorescence

  • Solution: Increase blocking time/concentration; use specific blocking agents; include autofluorescence quenching steps; optimize washing protocols

• Inconsistent staining patterns:

  • Cause: Variable fixation, sample heterogeneity, antibody aggregation

  • Solution: Standardize fixation protocols; increase technical replicates; centrifuge antibody solution before use to remove aggregates

• Photobleaching during analysis:

  • Cause: FITC sensitivity to repeated light exposure

  • Solution: Minimize exposure time; use anti-fade mounting media; consider acquiring images from different fields rather than repeated acquisition from the same field

• Cross-reactivity concerns:

  • Cause: Antibody binding to non-target epitopes

  • Solution: Include appropriate controls (knockout/knockdown samples); validate specificity using alternative detection methods

Published protocols for detecting PMCA4 in red blood cells demonstrate successful mitigation of these challenges through optimized permeabilization with saponin, effective blocking with specialized buffers, and careful calibration of microscopy parameters .

How can researchers validate specific binding and optimize conditions for ATP2B4 Antibody, FITC conjugated?

To ensure specific binding and optimal experimental conditions for ATP2B4 Antibody, FITC conjugated, researchers should implement a systematic validation approach:

• Epitope specificity confirmation:

  • Compare staining patterns with antibodies targeting different epitopes of ATP2B4

  • Perform peptide competition assays using the immunizing peptide (e.g., recombinant ATP2B4 protein fragments)

  • Verify results using genetic models with altered ATP2B4 expression (knockdown/knockout/overexpression)

• Antibody titration matrix:

  • Create a dilution series (e.g., 1:100, 1:200, 1:400, 1:800)

  • Test each dilution with varying incubation times and temperatures

  • Determine optimal signal-to-noise ratio conditions

• Multi-method validation:

  • Complement immunofluorescence findings with alternative techniques

  • Consider Western blotting, enzyme assays like BCIP/NBT substrate reaction , or mass spectrometry

  • Correlate protein detection with mRNA expression using qRT-PCR

• Functional validation:

  • Assess calcium transport activity in sorted cell populations based on ATP2B4 staining intensity

  • Correlate staining patterns with functional responses to calcium perturbation

  • Use pharmacological inhibitors of PMCA4 to confirm specificity of observed effects

• Cross-platform verification:

  • Compare results between flow cytometry and microscopy

  • Verify membrane localization using subcellular fractionation followed by Western blotting

  • Correlate findings with super-resolution microscopy for precise localization

Research examining PMCA4 in multiple myeloma has successfully employed multiple validation approaches, including complementary detection methods (immunofluorescence and Western blotting) and correlation with functional outcomes (calcium content in RBCs), establishing robust protocols for ATP2B4 antibody applications .

How can ATP2B4 Antibody, FITC conjugated be used to investigate calcium dysregulation in hematological disorders?

ATP2B4 Antibody, FITC conjugated provides a valuable tool for investigating calcium dysregulation in hematological disorders, particularly given the importance of PMCA4 in red blood cell calcium homeostasis:

• Comparative expression analysis:

  • Collect blood samples from patients with various hematological disorders and healthy controls

  • Prepare RBC smears and label with ATP2B4 Antibody, FITC conjugated

  • Quantify fluorescence intensity using standardized microscopy settings

  • Analyze differences in PMCA4 expression and distribution patterns

• Correlation with disease parameters:

  • In conditions like multiple myeloma, correlate PMCA4 expression with:

    • Disease stage and progression

    • Calcium levels in serum

    • Presence of osteolytic lesions

    • Treatment response

• RBC morphology and function correlation:

  • Combine ATP2B4 staining with morphological assessment

  • Look for correlations between PMCA4 expression and RBC abnormalities like rouleaux formation (stacking of RBCs), which has been observed in MM

  • Assess RBC functional parameters such as deformability and oxygen transport capacity

• Mechanistic investigation:

  • Explore regulatory mechanisms affecting ATP2B4 expression

  • Investigate miRNA regulation (e.g., miR-4261) in patient samples

  • Assess exosomal transfer of regulatory molecules between malignant cells and RBCs

Research has demonstrated that PMCA4 expression is significantly reduced in RBCs of multiple myeloma patients, correlating with calcium overload and potentially contributing to disease pathophysiology . This approach can be extended to other hematological disorders where calcium dysregulation may play a role.

What insights can ATP2B4 localization provide about gene variants and their functional impact?

ATP2B4 Antibody, FITC conjugated enables detailed analysis of PMCA4 localization, which can provide critical insights into the functional impact of gene variants:

• Expression and localization comparison:

  • Express wild-type ATP2B4 and variant forms (e.g., C3152A, C31528T, G3346A) in model cell systems

  • Use ATP2B4 Antibody, FITC conjugated to visualize protein localization

  • Quantify membrane vs. cytoplasmic distribution

  • Identify any aggregation, mislocalization, or altered membrane incorporation

• Structure-function analysis:

  • Correlate variants in specific domains (transmembrane, cytoplasmic, regulatory) with localization patterns

  • Assess impact on protein-protein interactions at the membrane

  • Determine if variants affect incorporation into lipid rafts or specialized membrane domains

• Temporal dynamics assessment:

  • Perform live-cell imaging using expression systems with fluorescently-tagged ATP2B4 variants

  • Measure protein turnover rates and membrane stability

  • Assess response to calcium flux in different variants

• Correlation with clinical phenotypes:

  • For variants identified in patients (such as those with Bihormonal Adrenocorticotropic Hormone-secreting Pituitary Adenoma (BHA) ), correlate localization patterns with clinical presentation

  • Determine if specific localization changes correlate with disease severity or manifestations

Research has identified rare gene variants in ATP2B4 in patients with BHA, which belongs to the same family of Ca-ATPases as ATP2B3, a gene previously implicated in this condition . Using ATP2B4 Antibody, FITC conjugated to study localization patterns of these variants can provide insights into pathogenic mechanisms and potential therapeutic approaches.

How can quantitative analysis of ATP2B4 distribution be performed in tissue samples?

Quantitative analysis of ATP2B4 distribution in tissue samples using ATP2B4 Antibody, FITC conjugated requires systematic approaches:

• Tissue preparation and staining protocol:

  • For frozen sections: Fix with cold methanol or 4% paraformaldehyde

  • For paraffin sections: Perform heat-induced epitope retrieval

  • Block with appropriate serum (5-10% normal goat serum)

  • Apply ATP2B4 Antibody, FITC conjugated at optimized dilution

  • Counterstain with DAPI for nuclear visualization

  • Mount with anti-fade medium to preserve fluorescence

• Microscopy acquisition settings:

  • Standardize acquisition parameters (exposure time, gain, offset)

  • Take Z-stack images to capture full tissue thickness

  • Include scale bars for accurate size reference

  • Capture multiple fields per sample for representative analysis

• Quantification strategies:

  • Membrane intensity analysis:

    • Define membrane regions using automated algorithms or membrane markers

    • Measure fluorescence intensity along membrane segments

    • Calculate mean membrane intensity and coefficient of variation

  • Distribution pattern analysis:

    • Assess polarization indices (apical vs. basolateral distribution)

    • Quantify clustering using nearest neighbor analysis

    • Measure co-localization with other membrane proteins

  • Regional analysis:

    • For adrenal tissues, separate analysis by zones (as mentioned in search result )

    • For other tissues, define anatomical regions for comparative analysis

    • Normalize expression to appropriate housekeeping proteins

• Statistical analysis approaches:

  • Compare ATP2B4 expression across different tissue types or disease states

  • Correlate with clinical parameters or experimental conditions

  • Apply appropriate statistical tests based on data distribution

These approaches have been applied in various contexts, including analysis of ATP2B4/PMCA4 expression in adrenal tissues and blood cells , enabling quantitative comparison between normal and pathological states.

How can ATP2B4 Antibody, FITC conjugated be used to investigate the relationship between miRNAs and calcium transport proteins?

Recent research has revealed important relationships between microRNAs and calcium transport proteins like ATP2B4/PMCA4. ATP2B4 Antibody, FITC conjugated can be instrumental in exploring these regulatory networks:

• miRNA target validation:

  • Transfect cells with specific miRNAs (e.g., miR-4261) or antagomirs

  • Use ATP2B4 Antibody, FITC conjugated for flow cytometry or imaging

  • Quantify changes in PMCA4 expression at single-cell resolution

  • Correlate with functional calcium transport assays

• Mechanistic pathway analysis:

  • Perform time-course experiments after miRNA transfection

  • Track changes in ATP2B4 localization and expression

  • Combine with RNA-FISH to visualize miRNA and ATP2B4 mRNA in the same cells

  • Determine if effects are direct (translation inhibition) or indirect (transcriptional regulation)

• Exosomal transfer studies:

  • Isolate exosomes from cells (e.g., multiple myeloma cells)

  • Apply to recipient cells (e.g., RBCs) in Transwell systems

  • Use ATP2B4 Antibody, FITC conjugated to track changes in PMCA4 expression

  • Correlate with exosomal miRNA content

Research has demonstrated that exosomal miR-4261 can mediate calcium overload in RBCs by targeting ATP2B4, as confirmed through dual-luciferase assays that validated the direct interaction between miR-4261 and the 3'-UTR of ATP2B4 mRNA . The relationship was further substantiated by showing that miR-4261 mimics reduced both mRNA and protein levels of ATP2B4, with the most significant reduction observed 72 hours post-transfection .

What advanced imaging techniques can enhance the utility of ATP2B4 Antibody, FITC conjugated in research?

Advanced imaging techniques can significantly enhance the information obtained from ATP2B4 Antibody, FITC conjugated staining:

• Super-resolution microscopy:

  • Apply techniques like STORM, PALM, or STED to visualize ATP2B4 distribution below the diffraction limit

  • Analyze nanoscale clustering and organization in the membrane

  • Combine with other membrane proteins to assess co-organization in functional domains

• Live-cell imaging approaches:

  • Use expression systems with ATP2B4-FP fusions verified against antibody staining

  • Perform FRAP (Fluorescence Recovery After Photobleaching) to measure protein mobility

  • Track protein dynamics during calcium signaling events

• Correlative light and electron microscopy (CLEM):

  • Localize ATP2B4 using the FITC-conjugated antibody in light microscopy

  • Process the same sample for electron microscopy

  • Correlate protein localization with ultrastructural features

• Calcium imaging integration:

  • Combine ATP2B4 staining with calcium indicators

  • Perform ratio imaging to correlate local ATP2B4 concentration with calcium flux

  • Use computational approaches to map spatial relationships

• Expansion microscopy:

  • Apply physical expansion of samples after ATP2B4 staining

  • Achieve effective super-resolution with standard confocal microscopy

  • Preserve spatial relationships while enhancing visualization of membrane distribution

• Light-sheet microscopy:

  • Achieve rapid 3D imaging of ATP2B4 distribution in thick samples

  • Reduce photobleaching compared to confocal approaches

  • Enable long-term imaging of dynamic processes