EZR Antibody

What is Ezrin (EZR) and what are its key functions in cellular biology?

Ezrin (EZR) is a member of the ezrin-radixin-moesin (ERM) protein family that functions as a crucial link between the plasma membrane and the actin cytoskeleton. This ~81 kDa protein plays several critical roles:

• Acts as a protein-tyrosine kinase substrate in microvilli

• Required for the formation of microvilli and membrane ruffles on epithelial cells' apical pole

• Facilitates normal macropinocytosis in conjunction with PLEKHG6

• Regulates B cell receptor (BCR) signaling and tunes humoral immunity strength

• Contributes to membrane-cytoskeleton remodeling during cell activation

Research has demonstrated that ezrin deficiency can lead to significant immune dysfunction. A 2023 study identified a homozygous missense mutation (A129T) in the EZR gene that impairs adaptive immunity . Additionally, ezrin regulates the amplification of BCR signals by controlling lipid raft dynamics and BCR microcluster formation, directly impacting B cell activation strength and antibody production .

What types of EZR antibodies are available and how should researchers select the appropriate one?

Researchers can choose from several types of EZR antibodies based on their experimental needs:

When selecting an antibody:

• Consider the specific application (e.g., WB, IF, IHC)

• Determine required species reactivity (human, mouse, rat, etc.)

• Assess whether total EZR or phosphorylated forms are the target

• Review validation data for your specific application

For studying EZR phosphorylation dynamics, phospho-specific antibodies targeting Tyr146 or Tyr353 residues are available and have been validated in research settings . For membrane-cytoskeleton interaction studies, antibodies recognizing the FERM domain may be more appropriate.

What are the optimal dilution ratios and experimental conditions for different applications of EZR antibodies?

Optimal working dilutions and conditions vary by application and specific antibody:

Western Blotting:

• Monoclonal antibodies: 1:1000 dilution is standard

• Polyclonal antibodies: 1:500-1:2000 range

• Optimal protein loading: 10-30 μg of total protein

• Detection: HRP-conjugated secondary antibodies work well with ezrin detection

• Expected MW: 81 kDa (may vary slightly depending on cell type)

Immunohistochemistry:

• Paraffin sections: 1:50-1:100 dilution

• Antigen retrieval: Heat-induced epitope retrieval with basic pH buffer is recommended

• Blocking: 5% normal serum from the same species as the secondary antibody

• Visualization: Both DAB and fluorescent detection systems yield good results

Immunofluorescence/Immunocytochemistry:

• Recommended dilutions: 1:100-1:500

• Fixation: 4% paraformaldehyde for 10 minutes at room temperature

• Permeabilization: 0.1% Triton X-100 for 10 minutes

• Blocking: 5-10% normal serum for 45-60 minutes

Flow Cytometry:

• Fixed/permeabilized cells: 1:50 dilution

• Use appropriate isotype controls

Researchers should always conduct preliminary titration experiments to determine optimal conditions for their specific experimental system .

How can researchers validate the specificity of their EZR antibody?

Validation of EZR antibody specificity is critical for reliable results. Recommended methodologies include:

• Positive and negative control samples:

  • Positive controls: Human placenta tissue, HeLa cells, and C6 rat glioma cell lines have demonstrated reliable ezrin expression

  • Negative controls: Use tissue/cells known to have low or no ezrin expression

• Knockout/knockdown validation:

  • Compare antibody staining in EZR-deficient (Ez-def) samples versus wild-type

  • siRNA or shRNA knockdown of EZR can serve as valuable controls

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide before staining

  • Loss of signal indicates specificity, as demonstrated in UV-treated NIH-3T3 cells

• Detection of expected molecular weight:

  • Ezrin should appear at approximately 81 kDa on Western blots

  • Additional bands may indicate non-specific binding or degradation products

• Multiple antibody validation:

  • Use multiple antibodies targeting different epitopes

  • Consistent results across antibodies suggest specific detection

• Cross-application validation:

  • Consistent results across multiple applications (WB, IF, IHC) strengthen confidence in specificity

Enhanced validation techniques employed by some manufacturers include testing on various species and cell types to ensure cross-reactivity claims are accurate .

What methods can be used to study ezrin phosphorylation and how do phospho-specific antibodies aid this research?

Ezrin function is regulated by phosphorylation at specific residues. To study these modifications:

Phospho-specific antibody approaches:

• Use antibodies targeting specific phosphorylation sites:

  • Tyr146 phosphorylation: Mouse monoclonal antibodies (e.g., sc-166858)

  • Tyr353 phosphorylation: Rabbit polyclonal antibodies (e.g., 3144, Cell Signaling)

  • Thr566 (or non-phosphorylated state): Region-specific antibodies

Experimental methodologies:

• Stimulation time course studies:

  • Treat cells with appropriate stimuli (e.g., LPS for B cells, antigen receptor crosslinking)

  • Collect lysates at various time points

  • Analyze phosphorylation kinetics by Western blotting with phospho-specific antibodies

• Inhibitor approaches:

  • Use kinase inhibitors to block specific signaling pathways

  • Assess impact on ezrin phosphorylation status

• Mutational analysis:

  • Compare wild-type ezrin with phospho-null (T→A) or phospho-mimetic (T→D/E) mutants

  • Evaluate functional consequences of these mutations

• Phosphatase treatment controls:

  • Treat duplicate samples with lambda phosphatase

  • Loss of signal with phospho-specific antibodies confirms specificity

Research has demonstrated that threonine dephosphorylation of ezrin leads to breakdown of barriers that normally limit B-cell receptor diffusion, while subsequent rephosphorylation reforms ERM-based corrals , highlighting the dynamic nature of ezrin regulation by phosphorylation.

How does ezrin regulate B cell activation and what methodologies are best for studying this mechanism?

Ezrin plays a critical role in regulating B cell activation through several mechanisms:

Key findings from research:

• Ezrin regulates the size and stability of B cell receptor (BCR) microclusters

• In ezrin-deficient B cells, BCR microclusters are larger and more stable, recruiting more tyrosine-phosphorylated proteins

• Ezrin absence leads to stronger activation of proximal signaling mediators and the ERK pathway

• Ezrin-deficient B cells show increased proliferation and differentiation into antibody-secreting cells

• Mice lacking ezrin in B cells exhibit magnified humoral responses to immunization

Recommended methodologies:

• Single-molecule localization microscopy (STORM):

  • For detailed analysis of BCR microcluster dynamics

  • Measures BCR diffusion coefficients and cluster sizes

  • Can reveal ezrin's role in confining BCR movement

• Live cell TIRF microscopy:

  • Visualizes real-time formation of BCR microclusters

  • Tracks ezrin redistribution during B cell activation

  • Enables measurement of cluster intensity and trajectory

• Phospho-flow cytometry:

  • Quantifies signaling pathway activation (ERK, calcium flux)

  • Allows single-cell analysis of population heterogeneity

  • Can be combined with surface marker staining to identify B cell subsets

• Ex vivo functional assays:

  • CFSE labeling to track proliferation of ezrin-deficient versus wild-type B cells

  • ELISPOT to quantify antibody-secreting cell differentiation

  • LPS + anti-IgM stimulation protocols reveal ezrin's influence on B cell activation

• In vivo immunization models:

  • T-dependent (e.g., NP-CGG with LPS adjuvant) and T-independent (e.g., NP-Ficoll) immunizations

  • Measurement of antigen-specific antibody responses over time

  • Analysis of different antibody isotypes (IgM, IgG1, IgG2a, etc.)

These approaches have revealed that ezrin limits lipid raft dynamics and BCR microcluster growth during the earliest phases of B cell activation, providing a novel mechanism for regulation of BCR signal amplification .

What are the best methods for using EZR antibodies in immunofluorescence and confocal microscopy?

Successful immunofluorescence detection of ezrin requires careful attention to sample preparation and staining protocols:

Sample preparation optimization:

• Fixation:

  • 4% paraformaldehyde for 10 minutes at room temperature preserves ezrin structure

  • Avoid methanol fixation which can disrupt membrane proteins

• Permeabilization:

  • 0.1% Triton X-100 in PBS for 10 minutes provides optimal access to ezrin epitopes

  • For membrane-associated ezrin, gentler permeabilization with 0.05% saponin may better preserve localization

• Blocking:

  • 5% donkey serum (or serum matching secondary antibody species)

  • Block for 45-60 minutes at room temperature

  • BSA (3-5%) can be used as an alternative

Staining protocol:

• Primary antibody incubation:

  • Dilute antibody in blocking buffer (1:100-1:200 for most preparations)

  • Incubate for 2 hours at room temperature or overnight at 4°C

• Secondary antibody selection:

  • Use highly cross-adsorbed secondary antibodies to minimize background

  • Fluorophore selection should consider spectral compatibility with other channels

  • Examples: Alexa Fluor 488, 557, or 637-conjugated antibodies have been successfully used

• Co-staining strategies:

  • For membrane-cytoskeleton interactions: Co-stain with phalloidin to visualize F-actin

  • For immune synapse studies: Co-stain with CD3 (T cells) or BCR components (B cells)

  • Nuclear counterstaining with DAPI aids in cellular orientation

Imaging considerations:

• Use confocal microscopy for precise subcellular localization

• For quantitative analysis of ezrin redistribution, standardize acquisition settings

• Z-stack acquisition can reveal the 3D distribution of ezrin, particularly at the apical surface

• Super-resolution techniques (STED, STORM) may be required to resolve ezrin-based membrane domains

In human colon stem cells, ezrin has been successfully visualized using sheep anti-human/mouse/rat ezrin antibody at 0.5 μg/mL, with specific staining localized to microtubules .

How can researchers troubleshoot common issues with EZR antibody detection in Western blotting?

Western blotting with EZR antibodies can present several challenges. Here are methodological solutions to common problems:

Issue 1: Weak or no signal

• Methodological solutions:

  • Increase antibody concentration (try 1:500 if 1:1000 yields weak signal)

  • Extend primary antibody incubation to overnight at 4°C

  • Ensure adequate protein loading (20-30 μg of total protein)

  • Use enhanced chemiluminescence (ECL) substrate with higher sensitivity

  • Verify transfer efficiency with reversible staining (Ponceau S)

  • Check ezrin expression level in your sample (replace with known positive control like human placenta)

Issue 2: Multiple bands or high background

• Methodological solutions:

  • Increase blocking stringency (5% BSA or milk for 1-2 hours)

  • Add 0.1% Tween-20 to antibody dilution buffer

  • Increase wash duration and number (5 × 5 minutes with TBS-T)

  • Reduce primary antibody concentration

  • Filter primary and secondary antibody solutions

  • Use freshly prepared buffers

  • Ensure membrane was completely blocked before antibody addition

Issue 3: Unexpected molecular weight

• Methodological solutions:

  • Expected MW for ezrin is approximately 81 kDa

  • Lower MW bands may indicate degradation: add protease inhibitors during lysis

  • Higher MW bands may indicate post-translational modifications or aggregation

  • Verify with reducing conditions (include DTT or β-mercaptoethanol)

  • Compare to literature: phospho-ezrin may migrate slightly higher

Issue 4: Inconsistent results between experiments

• Methodological solutions:

  • Standardize lysate preparation (consistent lysis buffer, protein determination method)

  • Include loading control (β-actin, β-tubulin) for normalization

  • Prepare larger antibody aliquots to avoid freeze-thaw cycles

  • Use the same membrane type and transfer protocol between experiments

  • Consider quantitative techniques like Simple Western™ for more consistent results

Validation approaches:

• Include both positive control (human placenta, HeLa, NIH-3T3 cells)

• Include negative control (ezrin-knockdown samples if available)

• Test with blocking peptide to confirm specificity

What is the role of ezrin in cancer progression and how can EZR antibodies be used to study this relationship?

Ezrin plays significant roles in cancer biology, particularly in processes related to metastasis and invasion:

Ezrin's roles in cancer:

• Facilitates formation of membrane structures required for cancer cell migration

• Contributes to signaling pathways promoting cancer cell survival

• Involved in epithelial-to-mesenchymal transition

• Associated with multiple neoplasms, carcinomas, and adenocarcinomas

Methodological approaches using EZR antibodies:

• Tissue microarray analysis:

  • IHC staining of tumor tissues with anti-EZR antibodies (1:50-1:100 dilution)

  • Comparison of ezrin expression between normal, primary tumor, and metastatic tissues

  • Correlation with clinical outcomes and pathological features

• Cell line models:

  • Western blot analysis of ezrin expression across cancer cell lines

  • Immunofluorescence to evaluate subcellular localization in aggressive vs. non-aggressive lines

  • Correlation with invasive and migratory capabilities

• Functional studies:

  • Ezrin knockdown/knockout approaches followed by:

    • Invasion assays (Boyden chamber, 3D matrix invasion)

    • Migration assays (wound healing, single-cell tracking)

    • Immunostaining to visualize changes in cell morphology and cytoskeletal organization

• Phosphorylation-specific analysis:

  • Use of phospho-specific antibodies (Tyr353, Tyr146) to assess activation state

  • Correlation of phosphorylation status with invasive properties

  • Treatment with kinase inhibitors to block ezrin activation

• In vivo metastasis models:

  • Injection of ezrin-manipulated cancer cells into nude mice

  • Analysis of tumor growth and metastatic spread

  • Immunohistochemical staining of primary and metastatic tumors

Research has demonstrated ezrin's relevance in various cancer types, with publications linking ezrin to neoplasms (>4 publications), carcinoma (>2 publications), adenocarcinoma and breast neoplasms . An animal study involving nude mice showed that knockdown of EZR-AS1 (a long non-coding RNA that regulates ezrin expression) affected tumor growth, suggesting therapeutic potential in targeting the ezrin pathway .

How do researchers quantitatively analyze EZR expression levels across different experimental conditions?

Accurate quantification of ezrin expression requires rigorous methodology and appropriate controls:

Western blot quantification methods:

• Densitometric analysis:

  • Capture images using a linear detection system (ChemiDoc Touch or similar)

  • Use analysis software (ImageJ, Image Lab) to measure band intensities

  • Normalize ezrin signal to loading control (β-actin, β-tubulin)

  • Express as fold-change relative to control condition

• Technical considerations:

  • Ensure exposure is within linear range (no saturated pixels)

  • Include standard curve of recombinant protein if absolute quantification is needed

  • Run all samples to be compared on the same gel when possible

  • Include biological replicates (minimum n=3) for statistical analysis

Flow cytometry quantification:

• Quantitative flow cytometry:

  • Use calibration beads with known antibody binding capacity

  • Calculate molecules of equivalent soluble fluorochrome (MESF)

  • Compare mean fluorescence intensity (MFI) across populations

  • Gate on relevant cell populations using forward/side scatter and surface markers

• Data analysis approach:

  • Subtract isotype control MFI from test sample MFI

  • Present as histogram overlays for visual comparison

  • Calculate percent positive cells using appropriate gating strategy

Immunofluorescence quantification:

• Image analysis protocols:

  • Acquire images using identical settings across all conditions

  • Measure mean fluorescence intensity within defined regions of interest

  • Subtract background from cell-free areas

  • Analyze multiple cells (>30) from multiple fields

• Advanced analysis:

  • Measure membrane-to-cytoplasm ratio to quantify translocation

  • Use colocalization analysis (Pearson's coefficient) for interaction studies

  • Employ 3D reconstruction for volumetric quantification

ELISA-based quantification:

• Sandwich ELISA approach:

  • Use commercially available ezrin ELISA kits

  • Create standard curve with purified ezrin

  • Ensure samples fall within the linear range of detection

  • Calculate concentration based on standard curve

A robust quantification workflow should include:

• Multiple technical replicates

• Appropriate statistical analysis (t-test, ANOVA with post-hoc tests)

• Presentation of data with error bars representing standard deviation or standard error

• Normalization to account for variations in cell number or protein content

Research examining ezrin's role in B cell activation employed these quantitative approaches to demonstrate increased BCR microcluster intensity in ezrin-deficient cells compared to wild-type controls .

What are the differences between studying ezrin in fixed versus live cell imaging, and which EZR antibodies are suitable for each approach?

Fixed and live cell imaging offer complementary insights into ezrin biology, each with distinct methodological considerations:

Fixed Cell Imaging:

Advantages:

• Higher signal-to-noise ratio

• Multiple antibody labeling possible

• Preserves structures for detailed analysis

• Compatible with super-resolution techniques

Recommended antibodies and methods:

• Most commercial anti-EZR antibodies work well for fixed samples

• Polyclonal antibodies (1:100-1:200) often provide stronger signals

• Fixation with 4% paraformaldehyde for 10 minutes preserves ezrin structure

• Permeabilization with 0.1% Triton X-100 allows antibody access to intracellular ezrin

Application examples:

• Immunofluorescence of ezrin in human colon stem cells using sheep anti-human/mouse/rat ezrin antibody (0.5 μg/mL)

• Co-staining with other proteins (e.g., chromogranin A) to establish contextual localization

• STORM imaging to measure precise BCR microcluster size and diffusion coefficients in relation to ezrin

Live Cell Imaging:

Advantages:

• Captures dynamic processes

• Reveals real-time protein redistribution

• Allows for tracking of protein movement

• Enables measurement of kinetic parameters

Recommended approaches:

• Fluorescent protein fusions (ezrin-GFP) rather than antibodies

• Anti-ezrin Fab fragments conjugated to fluorophores for some applications

• Live-cell compatible dyes for contextual staining (membrane, actin)

Application examples:

• TIRF microscopy to track ezrin redistribution during B cell activation

• Live imaging reveals two spatiotemporal phases of ezrin function in limiting BCR motility

• Ezrin-GFP fusion proteins have been used in nude mice studies

Comparative Analysis Methods:

When comparing fixed and live approaches:

• Validation strategy:

  • Confirm that ezrin-GFP localization matches antibody staining patterns

  • Ensure ezrin-GFP expression levels are physiologically relevant

  • Use minimally invasive imaging conditions for live cells

• Complementary insights:

  • Use live imaging to identify dynamic events

  • Follow with fixed cell imaging for molecular composition at specific timepoints

  • Correlate movement patterns from live imaging with structural details from fixed samples

• Technical considerations:

  • Account for fixation artifacts when interpreting structures

  • Consider photobleaching and phototoxicity in live cell experiments

  • Use fast acquisition for capturing rapid ezrin reorganization events

Research has demonstrated that fixed cell STORM imaging and live cell TIRF microscopy provide complementary insights into how ezrin regulates BCR diffusion and microcluster formation during B cell activation .

How does ezrin interact with other ERM family proteins, and what methodological approaches can distinguish between them?

Ezrin belongs to the ERM (Ezrin-Radixin-Moesin) family of proteins that share significant structural homology, making their distinction methodologically challenging:

Structural and functional relationships:

• ERM proteins share ~75% amino acid identity

• All contain a FERM domain, an α-helical domain, and a C-terminal ERM association domain (C-ERMAD)

• They exist in inactive (closed) and active (open) conformations

• Activation involves phosphorylation and PIP2 binding

• Family members may have both redundant and specific functions

Methodological approaches to distinguish ERM proteins:

• Antibody-based discrimination:

  • Use highly specific monoclonal antibodies targeting unique epitopes

  • Validate specificity against recombinant ezrin, radixin, and moesin

  • Consider peptide competition assays with ERM-specific peptides

  • Western blotting can separate ERM proteins based on slight MW differences

• Genetic manipulation approaches:

  • Generate knockout/knockdown models specific to each ERM protein

    • Example: Ez-def B cells showed phenotypes not fully compensated by moesin

  • Complement with re-expression of specific ERM members

  • Use siRNA/shRNA with validated specificity for individual ERM proteins

• Mass spectrometry-based identification:

  • Immunoprecipitate with anti-EZR antibody followed by MS analysis

  • Identify ERM-specific peptides to distinguish family members

  • Quantify relative abundance of each ERM protein

• Functional compensation analysis:

  • Determine whether moesin upregulation occurs in ezrin-deficient cells

  • Assess whether double knockdown produces more severe phenotypes

  • Test if expression of one ERM family member rescues deficiency of another

• Phosphorylation-specific analysis:

  • Use antibodies against phosphorylated forms of specific ERM proteins

  • Analyze differential phosphorylation kinetics among family members

  • Identify kinases with preferential activity toward specific ERM proteins

Research findings on ERM interactions:

Researchers investigating ezrin-specific functions should consider combined approaches, such as using specific antibodies alongside genetic manipulation to clearly distinguish the roles of individual ERM family members.

What are the differences between phospho-specific and non-phospho-specific EZR antibodies and when should each be used?

Phospho-specific and non-phospho-specific EZR antibodies serve distinct research purposes and require different experimental considerations:

Phospho-specific EZR antibodies:

Characteristics:

• Recognize ezrin only when phosphorylated at specific residues:

  • Tyr146 phosphorylation: Mouse monoclonal antibodies (e.g., sc-166858)

  • Tyr353 phosphorylation: Rabbit polyclonal antibodies (e.g., 3144, Cell Signaling)

  • Thr567 phosphorylation: Required for ezrin activation and membrane association

Optimal applications:

• Signaling pathway analysis:

  • Monitoring ezrin activation following receptor stimulation

  • Time-course studies of phosphorylation/dephosphorylation cycles

  • Identifying kinases and phosphatases that regulate ezrin

• Subcellular localization studies:

  • Tracking redistribution of activated ezrin to membrane structures

  • Correlating phosphorylation with functional outcomes

  • Distinguishing pools of active vs. inactive ezrin

• Methodological considerations:

  • Include phosphatase inhibitors during sample preparation

  • Use phosphatase treatment as negative control

  • Consider rapid fixation to preserve phosphorylation status

  • Blocking with BSA rather than milk (which contains phosphatases)

Non-phospho-specific EZR antibodies:

Characteristics:

• Recognize ezrin regardless of phosphorylation status

• Some may target regions around non-phosphorylation sites (e.g., T566)

• Detect total ezrin protein pool

Optimal applications:

• Expression level analysis:

  • Quantifying total ezrin protein abundance

  • Comparing ezrin expression across cell types or conditions

  • Normalizing phospho-ezrin signals to total ezrin

• Structural and interaction studies:

  • Immunoprecipitation of ezrin protein complexes

  • Immunohistochemical detection in tissue sections

  • Co-localization studies with potential binding partners

• Methodological considerations:

  • Selection of epitope may affect detection (N-terminal vs. C-terminal)

  • Consider conformational states (open vs. closed) that may mask epitopes

  • Standard sample preparation protocols are typically sufficient

Integrated research approaches:

For comprehensive analysis of ezrin biology, researchers often employ both types of antibodies in parallel:

• Phosphorylation ratio analysis:

  • Normalize phospho-ezrin signal to total ezrin

  • Accounts for changes in expression level vs. activation state

  • Western blots on parallel samples or sequential probing after stripping

• Activation state correlation:

  • Compare subcellular distribution of phospho-ezrin vs. total ezrin

  • Assess proportion of active ezrin in different cellular compartments

  • Dual immunofluorescence staining with different secondary antibodies

Research has shown that ezrin undergoes dephosphorylation and rephosphorylation cycles during B cell activation, contributing to the regulation of BCR mobility and signaling . This dynamic regulation would be impossible to study without phospho-specific antibodies used alongside antibodies detecting total ezrin.

How can researchers use EZR antibodies to investigate ezrin's role in immune synapse formation?

Ezrin plays critical roles in immune synapse formation, and antibody-based methodologies offer powerful approaches to investigate these functions:

Experimental design strategies:

• Immunological synapse visualization:

  • Create in vitro synapse models:

    • B cells + antigen-coated surfaces or antigen-presenting cells

    • T cells + anti-CD3/CD28-coated surfaces or antigen-presenting cells

  • Fix cells at different time points after synapse initiation

  • Immunostain with anti-EZR antibodies (1:100-1:200)

  • Co-stain for synapse markers (e.g., CD3, BCR components, F-actin)

  • Image using confocal or super-resolution microscopy

• Dynamic analysis in living cells:

  • Transfect cells with fluorescently-tagged ezrin

  • Compare localization patterns with fixed-cell antibody staining

  • Perform time-lapse imaging during synapse formation

  • Correlate ezrin dynamics with functional outcomes

• Molecular manipulation approaches:

  • Compare wild-type cells with ezrin-deficient cells

  • Rescue experiments with wild-type ezrin or phospho-mutants

  • Use phospho-specific antibodies to track ezrin activation state during synapse formation

Key methodological considerations:

• Sample preparation for optimal visualization:

  • Use supported lipid bilayers or cell couples for synapse formation

  • Precise timing of fixation to capture different synapse stages

  • Gentle fixation to preserve delicate membrane structures

  • Appropriate permeabilization to maintain synapse architecture

• Quantitative analysis methods:

  • Measure ezrin enrichment/depletion at synapse interface

  • Analyze colocalization with receptors and signaling molecules

  • Quantify changes in distribution following stimulation

  • Compare ezrin dynamics with functional outcomes (e.g., signaling strength)

• Controls and validation:

  • Include non-synapse-forming cell pairs as controls

  • Use phosphatase treatment to validate phospho-specific staining

  • Employ ezrin knockdown/knockout cells to confirm antibody specificity

  • Compare multiple antibodies targeting different ezrin epitopes

Research findings and applications:

Research has revealed that ezrin regulates B cell receptor dynamics during activation:

• Ezrin limits the growth and stability of BCR microclusters

• In ezrin-deficient B cells, BCR forms larger and more stable clusters that recruit more signaling molecules

• This regulation occurs during the earliest phases of BCR-mediated B cell activation

• The absence of ezrin results in a magnified humoral response to immunization

In T cells, dephosphorylation of ERM proteins reduces cellular rigidity, leading to more efficient T cell-antigen presenting cell conjugate formation . This suggests that ezrin plays a critical role in regulating the physical properties of lymphocytes during immune synapse formation.

What is the significance of ezrin in epithelial cell biology and how can researchers best study this using EZR antibodies?

Ezrin plays crucial roles in epithelial cell biology, particularly in apical membrane organization and specialized structure formation:

Key functions in epithelial cells:

• Required for formation of microvilli and membrane ruffles on the apical pole

• Serves as a linker between plasma membrane proteins and the actin cytoskeleton

• Contributes to cell polarity establishment and maintenance

• Facilitates proper organization of membrane protein complexes

• Regulates cell-cell and cell-matrix adhesions

Recommended methodological approaches:

• Polarized epithelial cell models:

  • Culture cells on Transwell filters to establish apical-basolateral polarity

  • Allow full polarization (typically 7-14 days depending on cell type)

  • Perform immunostaining for ezrin from both apical and basolateral compartments

  • Use confocal microscopy with z-stack acquisition to visualize 3D distribution

• Optimal immunofluorescence methods:

  • Fixation: 4% paraformaldehyde to preserve membrane structures

  • Permeabilization: Gentle methods (0.1% Triton X-100 or 0.05% saponin)

  • Primary antibodies: 1:100-1:200 dilution of anti-EZR antibodies

  • Co-staining markers: F-actin (phalloidin), apical markers (e.g., villin), basolateral markers

• Functional manipulation studies:

  • siRNA/shRNA knockdown of ezrin in epithelial cells

  • Rescue experiments with wild-type or mutant ezrin

  • Live imaging of microvilli formation using ezrin-GFP

  • Correlation of ezrin distribution with epithelial barrier function

• Tissue-based analyses:

  • Immunohistochemistry on normal epithelial tissues

    • Human stomach: ezrin localizes to cell surface of epithelial cells in gastric glands

    • Use 1 μg/ml antibody concentration with heat-induced epitope retrieval

  • Compare ezrin distribution across different epithelial tissues

  • Assess changes in disease states (inflammation, metaplasia, dysplasia)

Advanced techniques and applications:

• Super-resolution microscopy:

  • For detailed analysis of ezrin organization within microvilli

  • Resolves individual microvilli structure beyond diffraction limit

  • Enables precise colocalization with other microvillar components

• Proximity ligation assay (PLA):

  • Identifies molecular interactions between ezrin and binding partners

  • Enables in situ detection of protein-protein interactions

  • Provides quantitative data on interaction frequency

• Calcium switch assays:

  • Monitor ezrin redistribution during epithelial junction assembly/disassembly

  • Correlate with establishment of polarity and barrier function

  • Track phosphorylation status using phospho-specific antibodies

• 3D organoid models:

  • Study ezrin in more physiologically relevant 3D structures

  • Examine role in lumen formation and epithelial organization

  • Compare normal versus disease-specific organoids

Research has demonstrated that ezrin is specifically localized to the apical surface of epithelial cells in gastric glands , highlighting its importance in establishing and maintaining apical specializations in different epithelial tissues.

How do experimental conditions affect EZR antibody performance and what optimization strategies are recommended?

Multiple experimental variables can significantly impact EZR antibody performance across different applications. Optimization strategies should address these factors:

Sample preparation factors and optimization:

• Fixation method:

  • Impact: Affects epitope preservation and accessibility

  • Optimization:

    • For immunofluorescence: 4% paraformaldehyde for 10 minutes at room temperature

    • For flow cytometry: 1-2% paraformaldehyde

    • Avoid methanol fixation which can disrupt membrane-associated ezrin

    • Test multiple fixation protocols if signal is suboptimal

• Protein extraction buffers:

  • Impact: Influences protein solubility and epitope preservation

  • Optimization:

    • Use RIPA buffer containing protease inhibitors for most applications

    • Include phosphatase inhibitors when studying phosphorylated forms

    • Sonication helps solubilize membrane-associated proteins

    • Avoid harsh detergents that may denature the epitope

• Antigen retrieval methods:

  • Impact: Critical for IHC applications, especially in formalin-fixed tissues

  • Optimization:

    • For paraffin sections: Heat-induced epitope retrieval with basic buffer (pH 9.0)

    • Optimize retrieval time (typically 10-20 minutes)

    • Test both citrate (pH 6.0) and Tris-EDTA (pH 9.0) buffers

Antibody application optimization:

• Dilution optimization:

  • Impact: Affects signal-to-noise ratio and specificity

  • Optimization:

    • Perform titration experiments: Test 2-3 dilutions above and below recommended range

    • Western blot: 1:500-1:2000 range

    • IHC/IF: 1:50-1:200 range

    • Document optimal conditions for each application and sample type

• Incubation conditions:

  • Impact: Influences antibody binding efficiency and background

  • Optimization:

    • Temperature: 4°C overnight vs. room temperature 1-2 hours

    • Use gentle agitation to ensure even antibody distribution

    • For primary antibodies, longer incubation at lower temperature often improves specificity

• Blocking optimization:

  • Impact: Reduces non-specific binding and background

  • Optimization:

    • Test different blocking agents (5% BSA, 5% normal serum, commercial blockers)

    • For phospho-specific antibodies, avoid milk (contains phosphatases)

    • Extend blocking time (1-2 hours) for problematic samples

Detection system considerations:

• Secondary antibody selection:

  • Impact: Affects signal amplification and specificity

  • Optimization:

    • Use highly cross-adsorbed secondary antibodies

    • Match host species to primary antibody (e.g., anti-rabbit for rabbit primaries)

    • For IF, select bright fluorophores (Alexa Fluor dyes) with appropriate spectral properties

• Signal amplification:

  • Impact: Enhances detection of low-abundance targets

  • Optimization:

    • For IHC: Consider polymer-based detection systems (VisUCyte™ HRP Polymer)

    • For WB: Use enhanced chemiluminescence substrates for increased sensitivity

    • For IF: Try tyramide signal amplification for weak signals

Evidence-based recommendations:

In research applications, successful detection of ezrin has been achieved using:

• For IHC in human stomach: 1 μg/ml sheep anti-ezrin with heat-induced epitope retrieval

• For IF in human colon stem cells: 0.5 μg/mL sheep anti-ezrin with NorthernLights™ 557-conjugated secondary antibody

• For WB of human/mouse placenta: 0.2 μg/mL sheep anti-ezrin antibody under reducing conditions

Systematic optimization along these parameters will ensure reliable and reproducible results across different experimental contexts.

What approaches can researchers use to study ezrin's interactions with the actin cytoskeleton?

Ezrin's primary function involves linking plasma membrane proteins to the actin cytoskeleton. Several methodological approaches can effectively investigate these interactions:

Biochemical interaction methods:

• Co-immunoprecipitation:

  • Immunoprecipitate ezrin using specific antibodies (IP dilution 1:50)

  • Western blot for co-precipitated actin and other binding partners

  • Consider chemical crosslinking to stabilize transient interactions

  • Use appropriate lysis conditions to preserve protein-protein interactions

• Proximity ligation assay (PLA):

  • Visualize direct protein-protein interactions in situ

  • Use anti-ezrin antibody paired with anti-actin antibody

  • Quantify interaction points in different cellular compartments

  • Compare interaction frequency under different stimulation conditions

• GST pull-down assays:

  • Express GST-tagged ezrin domains (particularly the C-terminal actin-binding domain)

  • Incubate with cell lysates or purified actin

  • Analyze binding by Western blot or mass spectrometry

  • Compare wild-type with mutant ezrin versions

Microscopy-based approaches:

• Co-localization analysis:

  • Immunostain cells with anti-EZR antibodies (1:100-1:200)

  • Co-stain with phalloidin to visualize F-actin

  • Image using confocal microscopy

  • Quantify co-localization using Pearson's or Manders' coefficients

• Live-cell dynamics:

  • Express fluorescently tagged ezrin (ezrin-GFP) and actin (actin-mCherry)

  • Perform time-lapse imaging during cellular processes (migration, spreading)

  • Track coordinated movements of ezrin and actin

  • Measure temporal relationships between ezrin recruitment and actin polymerization

• Super-resolution microscopy:

  • Use STORM or STED microscopy for nanoscale resolution

  • Analyze precise spatial relationships between ezrin and actin filaments

  • Quantify distances and organizational patterns beyond diffraction limit

  • Has been successfully used to study BCR microcluster dynamics in relation to ezrin

Functional manipulation studies:

• Domain mutation analysis:

  • Express ezrin with mutations in actin-binding domains

  • Assess impact on cellular processes (microvilli formation, migration)

  • Compare localization patterns with wild-type ezrin

  • Correlate with changes in actin organization

• Pharmacological approaches:

  • Treat cells with actin-disrupting drugs (latrunculin, cytochalasin D)

  • Examine consequences for ezrin localization and phosphorylation

  • Use jasplakinolide to stabilize actin filaments

  • Assess effects on ezrin-dependent cellular processes

• FRAP (Fluorescence Recovery After Photobleaching):

  • Measure dynamic exchange between cytoplasmic and membrane-associated ezrin

  • Compare recovery kinetics at actin-rich versus actin-poor regions

  • Determine how actin binding influences ezrin mobility

  • Test how activation state affects exchange rates

Research findings and applications:

Research has shown that ezrin plays critical roles in membrane-cytoskeleton remodeling during B cell activation . In epithelial cells, ezrin is required for the formation of apical microvilli , structures that depend on proper actin organization. These studies demonstrate how ezrin serves as a dynamic link between membrane components and the underlying actin cytoskeleton, with important functional consequences for cellular architecture and signaling.

How can researchers use EZR antibodies to investigate the role of ezrin in different cell types and tissues?

Ezrin functions vary across different cell types and tissues, requiring tailored experimental approaches to elucidate its context-specific roles:

Immune cells:

• B lymphocytes:

  • Key ezrin functions: Regulates BCR microcluster formation and signaling

  • Methodological approaches:

    • Flow cytometry with phospho-specific EZR antibodies to track activation

    • STORM/TIRF microscopy to visualize BCR-ezrin interactions

    • Ex vivo stimulation assays (anti-IgM, LPS) comparing wild-type and ezrin-deficient cells

    • Measure antibody production using ELISPOT after ezrin manipulation

• T lymphocytes:

  • Key ezrin functions: Regulates immune synapse formation, T cell rigidity

  • Methodological approaches:

    • Study ezrin localization during T cell-APC conjugate formation

    • Track ezrin phosphorylation during T cell activation

    • Analyze T cell subpopulations using flow cytometry and ezrin expression

    • Assess impact of ezrin deficiency on T cell functionality

Epithelial tissues:

• Gastric epithelium:

  • Key ezrin functions: Forms apical microvilli, regulates acid secretion

  • Methodological approaches:

    • IHC using 1 μg/ml anti-ezrin antibody on human stomach sections

    • Co-stain with epithelial markers to define ezrin's subcellular localization

    • Compare ezrin distribution in normal versus pathological samples

    • Correlate with gastric acid secretion pathway components

• Intestinal epithelium:

  • Key ezrin functions: Microvilli formation, barrier function

  • Methodological approaches:

    • Immunofluorescence on colon stem cells using 0.5 μg/mL anti-ezrin antibody

    • Study ezrin during intestinal epithelial differentiation

    • Analyze ezrin-dependent microvilli in intestinal organoids

    • Correlate ezrin expression with barrier function markers

Specialized tissues:

• Placenta:

  • Well-established ezrin expression: Validated as positive control tissue

  • Methodological approaches:

    • Western blot using 0.2 μg/mL anti-ezrin antibody

    • Compare human and mouse placental ezrin expression

    • Study ezrin's role in trophoblast function and placental development

• Neural tissues:

  • Key ezrin functions: Neuronal growth, glial function

  • Methodological approaches:

    • IHC/IF on brain sections using optimized antigen retrieval

    • Study ezrin in C6 rat glioma cell lines as established model

    • Compare ezrin expression across neural cell types

    • Investigate ezrin in neuronal growth cones and axon guidance

Comparative analysis strategies:

• Multi-tissue expression profiling:

  • Use validated anti-EZR antibodies for Western blot across tissue panel

  • Normalize expression to appropriate housekeeping proteins

  • Create tissue-specific expression profiles for ezrin

  • Correlate with tissue-specific functions

• Developmental analysis:

  • Track ezrin expression during differentiation of stem cells

    • Example: Human colon stem cells differentiating into ascending colon

  • Compare ezrin localization between progenitor and mature cell types

  • Investigate phosphorylation status changes during differentiation

• Pathological comparisons:

  • Compare ezrin expression/localization in normal versus diseased tissues

  • Study ezrin in inflammation and cancer models

  • Assess correlation with disease progression markers

  • Evaluate potential as biomarker for specific conditions

Technical considerations:

• Tissue-specific optimization:

  • Adapt fixation protocols to tissue type (duration, fixative composition)

  • Optimize antigen retrieval methods for each tissue

  • Adjust antibody concentration based on ezrin abundance in target tissue

  • Select appropriate positive control tissues (placenta, stomach)

• Validation approaches:

  • Confirm antibody specificity in each new tissue/cell type

  • Use multiple antibodies targeting different ezrin epitopes

  • Include ezrin-deficient controls when possible

  • Verify with independent techniques (gene expression, mass spectrometry)

These comprehensive approaches allow researchers to investigate the diverse and context-specific functions of ezrin across different biological systems.

What are the current limitations of EZR antibodies in research and how can researchers address them?

Despite their widespread use, EZR antibodies present several limitations that researchers should be aware of and address through appropriate methodological strategies:

Antibody specificity challenges:

• Cross-reactivity with other ERM proteins:

  • Limitation: Ezrin shares significant homology with radixin and moesin

  • Mitigation strategies:

    • Verify specificity using ERM knockout/knockdown controls

    • Use monoclonal antibodies targeting unique epitopes

    • Confirm results with multiple antibodies targeting different regions

    • Validate in systems with known ERM expression profiles

• Conformational state detection:

  • Limitation: Many antibodies cannot distinguish between active (open) and inactive (closed) ezrin

  • Mitigation strategies:

    • Use phospho-specific antibodies (Tyr146, Tyr353, Thr567) for active ezrin

    • Combine with functional assays that assess ezrin activity

    • Supplement with biochemical assays of ezrin activation state

    • Consider conformation-specific antibodies when available

• Post-translational modification interference:

  • Limitation: Modifications may mask antibody epitopes

  • Mitigation strategies:

    • Use multiple antibodies targeting different regions

    • Consider the impact of sample preparation on modifications

    • Include appropriate controls (phosphatase treatment, mutation of modification sites)

Technical limitations:

• Fixation-dependent epitope accessibility:

  • Limitation: Some epitopes are sensitive to fixation methods

  • Mitigation strategies:

    • Test multiple fixation protocols (PFA, methanol, acetone)

    • Optimize fixation duration and concentration

    • Explore various antigen retrieval methods for IHC/ICC

    • Compare native versus denatured detection systems

• Quantitative accuracy:

  • Limitation: Antibody binding may not be strictly proportional to protein amount

  • Mitigation strategies:

    • Include standard curves with recombinant protein

    • Use multiple antibodies and average results

    • Validate with orthogonal methods (mass spectrometry)

    • Employ spike-in controls to assess recovery efficiency

• Reproducibility between lots:

  • Limitation: Batch-to-batch variation, especially with polyclonal antibodies

  • Mitigation strategies:

    • Use monoclonal antibodies when possible

    • Purchase sufficient quantity of single lot for complete study

    • Include reference samples to normalize between experiments

    • Document lot numbers and perform bridging studies when changing lots

Application-specific limitations:

• Live-cell imaging limitations:

  • Limitation: Most antibodies require fixation and permeabilization

  • Mitigation strategies:

    • Use fluorescently-tagged ezrin constructs for live imaging

    • Consider Fab fragments for some applications

    • Validate live imaging findings with fixed-cell antibody staining

    • Use membrane-permeable small molecule probes when available

• Spatial resolution constraints:

  • Limitation: Standard microscopy may not resolve fine ezrin structures

  • Mitigation strategies:

    • Employ super-resolution techniques (STORM, STED)

    • Use deconvolution to improve conventional microscopy

    • Consider electron microscopy with immunogold labeling

    • Combine with proximity assays for interaction studies

Advanced alternative approaches:

• Genetic tagging strategies:

  • CRISPR/Cas9 knock-in of fluorescent or epitope tags

  • Ensures physiological expression levels

  • Enables live cell imaging without antibodies

  • Requires validation against antibody-based detection

• Proximity labeling methods:

  • BioID or APEX2 fusion proteins to identify interaction partners

  • Less dependent on antibody accessibility

  • Captures transient interactions

  • Complements traditional antibody-based co-IP approaches

• Mass cytometry (CyTOF):

  • Metal-conjugated antibodies for single-cell analysis

  • Eliminates autofluorescence issues

  • Enables simultaneous detection of many proteins

  • Provides quantitative data on ezrin in cellular subpopulations

These strategies can help researchers overcome the current limitations of EZR antibodies while maximizing the reliability and validity of their experimental findings.

How can researchers effectively interpret contradictory results when using different EZR antibodies?

Sources of contradictory results:

• Epitope-specific differences:

  • Different antibodies recognize distinct regions of ezrin

  • Epitopes may be differentially accessible depending on:

    • Protein conformation (active/inactive states)

    • Protein-protein interactions masking specific regions

    • Post-translational modifications affecting epitope accessibility

• Methodology-dependent discrepancies:

  • Sample preparation differences (fixation, extraction buffers)

  • Application-specific variables (reduced vs. non-reduced conditions for WB)

  • Detection system sensitivities (chromogenic vs. fluorescent)

  • Quantification approaches (densitometry methods, background subtraction)

• Antibody quality and specificity issues:

  • Cross-reactivity with other ERM family proteins

  • Batch-to-batch variation, especially in polyclonal antibodies

  • Non-specific binding to unrelated proteins

  • Primary vs. secondary antibody compatibility

Systematic resolution strategies:

• Comprehensive antibody validation:

  • Methodological approach:

    • Test multiple antibodies in parallel under identical conditions

    • Include positive and negative control samples

    • Use ezrin-knockdown/knockout samples as definitive controls

    • Perform peptide competition assays to confirm specificity

  • Implementation example:

    • Compare results from monoclonal (e.g., ZooMAb) and polyclonal antibodies

    • Validate across multiple applications (WB, IF, IHC)

    • Document specific conditions where discrepancies occur

• Orthogonal technique verification:

  • Methodological approach:

    • Confirm findings using antibody-independent methods

    • Correlate protein detection with mRNA expression

    • Use genetic approaches (tagged ezrin, CRISPR/Cas9 editing)

    • Apply mass spectrometry for definitive protein identification

  • Implementation example:

    • Verify Western blot results with RT-qPCR for EZR expression

    • Compare antibody staining with ezrin-GFP localization

    • Validate interaction findings with proximity ligation assays

• Epitope mapping and accessibility analysis:

  • Methodological approach:

    • Determine exact epitopes recognized by each antibody

    • Test antibodies against ezrin fragments/domains

    • Evaluate accessibility in different experimental conditions

    • Consider structural information about ezrin conformation

  • Implementation example:

    • Compare N-terminal vs. C-terminal targeting antibodies

    • Test sensitivity to phosphorylation status

    • Evaluate performance in native vs. denatured conditions

    • Use structural biology information to interpret accessibility

• Standardized reporting framework:

  • Methodological approach:

    • Document all experimental variables systematically

    • Record detailed antibody information (source, catalog number, lot, dilution)

    • Report both positive and negative findings

    • Acknowledge limitations and potential confounding factors

  • Implementation example:

    • Create detailed antibody validation profiles for each application

    • Document specific conditions where each antibody performs optimally

    • Maintain laboratory records of antibody performance across experiments

Integration and interpretation guidelines:

• Hierarchical evidence evaluation:

  • Weight results based on antibody validation quality

  • Prioritize findings confirmed by multiple antibodies

  • Consider findings from knockout/knockdown controls as gold standard

  • Evaluate consistency with established ezrin biology

• Context-dependent interpretation:

  • Consider biological context when assessing discrepancies

  • Different cell types may have different ezrin conformations or interactions

  • Activation state may affect epitope accessibility

  • Treatment conditions may alter post-translational modifications

• Quantitative concordance analysis:

  • Analyze trends rather than absolute values

  • Determine if antibodies show similar directional changes

  • Consider relative changes rather than absolute quantification

  • Apply appropriate statistical methods to evaluate significance of differences