CORO1B Antibody

What is CORO1B and what cellular functions does it regulate?

CORO1B (Coronin 1B) is an actin-binding protein that plays a critical role in controlling actin networks, particularly at classical lamellipodia. This 54 kDa protein (calculated molecular weight) is observed at approximately 68 kDa in gel electrophoresis experiments . CORO1B localizes to the plasma membrane where it regulates lamellipodia formation through interactions with the actin cytoskeleton . When phosphorylated, CORO1B loses its ability to bind the ARP2/3 complex, thereby reducing its actin nucleation inhibitory function . In endothelial cells, CORO1B has been found to control actin dynamics at cell-cell junctions and influence the assembly of endothelial networks, suggesting its importance in vascular biology . CORO1B also fine-tunes ROCK-signaling pathways to regulate myosin activity, further impacting cellular processes dependent on actin cytoskeleton reorganization .

What experimental applications are CORO1B antibodies validated for?

CORO1B antibodies have been validated for multiple experimental applications critical to molecular and cellular biology research. The most commonly validated applications include:

Researchers should note that optimal dilutions may vary depending on the specific experimental system and should be determined empirically for each application . For immunohistochemistry applications, antigen retrieval methods significantly impact staining quality, with TE buffer (pH 9.0) being recommended, though citrate buffer (pH 6.0) may serve as an alternative .

How should researchers optimize antibody dilutions for different experimental systems?

Optimizing antibody dilutions is critical for achieving specific signal while minimizing background. For CORO1B antibodies, researchers should begin with the manufacturer's recommended dilution ranges (e.g., 1:1000-1:4000 for Western blot, 1:50-1:500 for IHC) and then perform titration experiments to determine the optimal concentration for their specific experimental system.

This optimization process should include:

• Testing a range of dilutions in a pilot experiment

• Including appropriate positive controls (e.g., HT-29 cells, MDA-MB-453 cells, NIH/3T3 cells, mouse/rat brain tissue for Western blot)

• Including negative controls (secondary antibody only, isotype controls)

• Evaluating signal-to-noise ratio at each concentration

• Assessing reproducibility of results at the selected dilution

The optimal dilution should provide clear, specific signal with minimal background and may vary depending on protein expression levels in different tissue/cell types. Sample-dependent optimization is particularly important, and researchers should check validation data galleries provided by antibody manufacturers for reference .

What storage and handling conditions ensure optimal CORO1B antibody performance?

Proper storage and handling of CORO1B antibodies are essential for maintaining their performance across multiple experiments. Based on manufacturer recommendations, researchers should:

• Store antibodies at -20°C where they remain stable for one year after shipment

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• For working solutions, store in small aliquots (although aliquoting is noted as unnecessary for -20°C storage of some formulations)

• Handle antibodies on ice when preparing dilutions for experiments

• Be aware of formulation specifics - CORO1B antibodies are typically provided in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Note that some preparations may contain 0.1% BSA which can impact certain applications

These storage recommendations ensure antibody stability and consistent performance across experiments, which is particularly important for longitudinal studies requiring reproducible results over time.

How does Coronin 1B localize at endothelial cell-cell junctions and what methods best visualize this localization?

Coronin 1B demonstrates a distinctive localization pattern at endothelial cell-cell junctions that differs from classical adherens junction proteins. Unlike VE-cadherin, which shows a continuous distribution along cell-cell contacts, CORO1B exhibits a discontinuous pattern at junctions . This localization can be optimally visualized through:

• Confocal immunofluorescence microscopy using primary antibodies against CORO1B and junction markers like VE-cadherin

• Triple staining with phalloidin to visualize F-actin structures simultaneously

• Spinning-disk confocal microscopy for live-cell imaging when using Coro1B-GFP constructs

For optimal visualization, researchers should perform immunostaining on subconfluent endothelial cell monolayers (e.g., HUVECs or HMECs) grown on gelatin-coated glass coverslips . The protocol should include:

• Fixation with 4% paraformaldehyde for 10 minutes

• Permeabilization with 0.1% Triton X-100 for 30 minutes

• Blocking with 1% bovine serum albumin, 0.1% Triton X-100 in PBS for 1 hour

• Overnight incubation with primary antibodies at 4°C

• Application of species-specific Alexa Fluor-coupled secondary antibodies

This approach reveals that CORO1B is particularly enriched at the leading edge of junction-associated intermittent lamellipodia (JAIL), where it colocalizes with F-actin but shows only partial colocalization with VE-cadherin .

What is the relationship between CORO1B and the integrin-linked kinase (ILK) pathway in endothelial cells?

The relationship between CORO1B and integrin-linked kinase (ILK) represents an important signaling node in endothelial cell function. Analysis of the CORO1B interactome has identified ILK as a novel CORO1B-associated protein . This interaction has several functional implications:

• CORO1B colocalizes with α-parvin, an ILK interactor, specifically at the leading edge of lamellipodia protrusions

• This colocalization suggests that CORO1B may participate in integrin signaling pathways that regulate actin cytoskeleton dynamics

• The CORO1B-ILK interaction potentially links cell-matrix adhesion signals to cell-cell junction remodeling in endothelial cells

This interaction can be studied through co-immunoprecipitation experiments using GFP-Trap beads with Coro1B-GFP expressing cells, followed by western blot analysis with antibodies against ILK1 . Researchers investigating this pathway should consider:

• Using both endogenous co-immunoprecipitation and overexpression systems to validate interactions

• Performing immunofluorescence studies to confirm colocalization in cellular contexts

• Designing functional studies to assess how disrupting this interaction affects endothelial cell behaviors

Understanding this relationship may provide insights into mechanisms coordinating cell-matrix and cell-cell adhesion during vascular development and homeostasis.

How can researchers effectively use CORO1B antibodies in studying endothelial tube formation and network assembly?

CORO1B plays a critical role in endothelial network formation, making it an important target for angiogenesis research. Studies have shown that depletion of CORO1B results in reduced network complexity, tube number, and tube length in matrigel tube formation assays . Researchers investigating this aspect should:

• Utilize CORO1B antibodies in combination with knockdown/knockout approaches to establish causal relationships

• Design experiments that incorporate both fixed-sample immunostaining and live-cell imaging

• Consider multiple parameters when assessing network formation:

  • Network complexity (number of branching points)

  • Total tube length

  • Tube diameter

  • Tube stability over time

For immunofluorescence studies in tube formation assays, researchers should modify standard protocols to account for the three-dimensional nature of the networks:

• Extend fixation time to ensure complete penetration into the matrigel structure

• Increase permeabilization duration to improve antibody access

• Use longer incubation periods for primary antibodies (up to 24-48 hours at 4°C)

• Consider clearing techniques to improve imaging depth in thicker structures

• Employ confocal z-stacks to capture the full three-dimensional organization

Correlating CORO1B localization with functional defects in network formation can provide insights into the specific mechanisms by which this protein influences endothelial morphogenesis and angiogenesis.

What approaches can validate CORO1B antibody specificity in different experimental systems?

Antibody validation is crucial for ensuring reliable experimental results. For CORO1B antibodies, several complementary approaches can be employed to verify specificity:

• Genetic knockout/knockdown controls: Compare staining patterns between wild-type cells and those with CORO1B knockdown or knockout. The specific signal should be significantly reduced or absent in the latter .

• Western blot analysis: Confirm that the antibody detects a single band at the expected molecular weight (observed at approximately 68 kDa for CORO1B despite a calculated molecular weight of 54 kDa) .

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide/protein before application to samples. This should abolish specific staining.

• Cross-reactivity testing: Verify reactivity across different species if working with non-human models. Current CORO1B antibodies show reactivity with human, mouse, and rat samples .

• Orthogonal detection methods: Compare localization patterns using alternative detection methods such as:

  • Multiple antibodies targeting different epitopes of CORO1B

  • Fluorescently tagged CORO1B constructs (e.g., Coro1B-GFP)

  • RNA expression analysis (in situ hybridization or single-cell RNA-seq)

These validation approaches should be documented and included in methodology sections of publications to strengthen the reliability of reported findings.

How can researchers effectively combine CORO1B antibody staining with live-cell imaging approaches?

Combining fixed-cell antibody staining with live-cell imaging provides complementary insights into CORO1B dynamics. Researchers can implement the following strategy:

• Correlative live-cell imaging and immunofluorescence:

  • Perform live-cell imaging of cells expressing fluorescent markers (e.g., Lifeact-mCherry to visualize F-actin dynamics)

  • Fix cells at specific timepoints or immediately after imaging

  • Perform immunostaining for CORO1B and other proteins of interest

  • Relocate the same cells/regions for imaging and correlate dynamic behaviors with protein localization

• Fluorescent protein tagging combined with antibody validation:

  • Generate cell lines expressing Coro1B-GFP using lentiviral transduction

  • Validate that the GFP signal colocalizes with anti-CORO1B antibody staining

  • Perform live-cell imaging to track CORO1B dynamics in real-time

  • Use spinning-disk confocal microscopy at 37°C and 5% CO₂ for optimal results

• Technical considerations:

  • Transfection efficiency can be improved using the MATra-A reagent with a magnet plate for difficult-to-transfect endothelial cells

  • For stable expression, selection with puromycin (10 μg/mL) can be applied 72 hours post-transduction

  • When designing fluorescent fusion proteins, consider both N- and C-terminal tagging to determine which least affects protein function

This integrated approach allows researchers to connect dynamic cellular processes with the molecular components involved, providing deeper insights into CORO1B function in living cells.

What are common technical challenges when using CORO1B antibodies and how can they be addressed?

Researchers working with CORO1B antibodies may encounter several technical challenges that can affect experimental outcomes. Here are common issues and recommended solutions:

• High background in immunofluorescence:

  • Increase blocking time (extend to 2+ hours)

  • Use alternative blocking agents (5% normal serum from secondary antibody species)

  • Increase washing steps duration and number

  • Optimize primary antibody dilution through titration experiments

  • Consider using different detection systems (e.g., tyramide signal amplification for low abundance targets)

• Weak or absent signal in Western blot:

  • Verify protein loading amount (CORO1B is moderately expressed in most cells)

  • Check transfer efficiency using reversible membrane staining

  • Consider longer exposure times or more sensitive detection methods

  • Optimize lysis conditions to ensure complete protein extraction

  • Remember CORO1B may appear at 68 kDa despite calculated MW of 54 kDa

• Inconsistent immunohistochemistry results:

  • Test different antigen retrieval methods (recommended TE buffer pH 9.0 or alternative citrate buffer pH 6.0)

  • Extend antigen retrieval time for difficult samples

  • Verify tissue fixation conditions (overfixation can mask epitopes)

  • Consider using amplification systems for low abundance targets

• Species cross-reactivity issues:

  • Verify antibody species reactivity (current antibodies react with human, mouse, rat)

  • For untested species, perform validation experiments before proceeding with full studies

  • Consider using multiple antibodies targeting different epitopes to confirm findings

Addressing these challenges through methodical optimization can significantly improve experimental outcomes and data reliability.

How should researchers interpret CORO1B localization in the context of actin dynamics and cell-cell junctions?

Interpreting CORO1B localization patterns requires understanding its relationship with actin structures and cell-cell junction components. Based on current research:

• Classical lamellipodia vs. junction-associated protrusions:

  • CORO1B localizes to the leading edge of classical lamellipodia in single cells and subconfluent monolayers

  • In cell-cell junctions, CORO1B shows a discontinuous pattern that differs from classical junction proteins like VE-cadherin

  • This distinctive localization suggests specialized functions in junction-associated actin dynamics

• Colocalization analysis considerations:

  • CORO1B partially colocalizes with VE-cadherin at cell-cell junctions

  • Strong colocalization with F-actin is observed specifically at the leading edge of junction-associated intermittent lamellipodia (JAIL)

  • Colocalization with α-parvin occurs at lamellipodia protrusions, indicating involvement in integrin signaling pathways

• Dynamic analysis through live imaging:

  • CORO1B is recruited to and operates at actin-driven membrane protrusions at cell-cell junctions

  • Its recruitment involves mechanisms requiring relaxation of the actomyosin cytoskeleton

  • Temporal analysis of CORO1B dynamics relative to junction proteins provides insights into causal relationships

• Functional context interpretation:

  • Depletion of CORO1B causes defects in the actin cytoskeleton and cell-cell junctions

  • These defects suggest that CORO1B's localization at junction protrusions is functionally significant

  • Researchers should correlate localization patterns with functional outcomes in their specific cell systems

When interpreting CORO1B localization data, researchers should consider both spatial (where it localizes) and temporal (when it is recruited) aspects to fully understand its role in cell function.

What controls are essential when studying CORO1B using immunological techniques?

Rigorous controls are fundamental to generating reliable data with CORO1B antibodies. Researchers should implement the following controls:

• Negative controls:

  • Secondary antibody only controls to assess non-specific binding

  • Isotype controls using non-specific primary antibodies of the same isotype (rabbit IgG for many CORO1B antibodies)

  • Samples with CORO1B knockdown/knockout to establish specificity

• Positive controls:

  • Cell types with known CORO1B expression (HT-29 cells, MDA-MB-453 cells, NIH/3T3 cells)

  • Tissue samples with established expression (mouse/rat brain tissue, mouse liver tissue)

  • Recombinant CORO1B protein as a Western blot standard

• Validation controls:

  • Peptide competition assays to confirm epitope specificity

  • Dual-labeling with different antibodies targeting distinct CORO1B epitopes

  • Correlation of protein detection with mRNA expression data

• Technical controls:

  • Loading controls for Western blots (housekeeping proteins)

  • Membrane staining to verify transfer efficiency

  • Internal positive controls within tissue sections showing expected staining patterns

• Treatment controls for functional studies:

  • Cytoskeleton disrupting agents (e.g., cytochalasin D) to verify actin-dependent localization

  • ROCK pathway inhibitors (e.g., Y-27632) to assess recruitment mechanisms dependent on actomyosin relaxation

  • Thrombin treatment to induce junction remodeling and observe CORO1B dynamics

Implementing these controls systematically ensures that observations related to CORO1B are specific and biologically relevant rather than technical artifacts.

How might CORO1B antibodies be utilized in studying vascular disorders and endothelial dysfunction?

Given CORO1B's critical role in endothelial cell function, particularly in cell-cell junction regulation and network formation , CORO1B antibodies offer significant potential for investigating vascular disorders:

• Vascular permeability studies:

  • Analyze CORO1B expression and localization in models of vascular leak

  • Correlate CORO1B distribution patterns with barrier integrity measurements

  • Investigate how inflammatory mediators alter CORO1B dynamics at endothelial junctions

• Angiogenesis research:

  • Examine CORO1B expression during different stages of vascular development

  • Compare CORO1B localization in normal versus pathological angiogenesis (tumor vasculature)

  • Investigate the relationship between CORO1B and key angiogenic signaling pathways

• Atherosclerosis investigations:

  • Assess CORO1B expression in endothelial cells at atheroprone versus atheroprotected vascular regions

  • Correlate CORO1B patterns with flow-induced endothelial adaptations

  • Investigate how oxidized lipids affect CORO1B-dependent cytoskeletal remodeling

• Therapeutic target validation:

  • Use CORO1B antibodies to monitor changes in protein expression/localization following experimental therapeutics

  • Evaluate CORO1B as a biomarker for endothelial dysfunction in tissue samples

  • Develop screening assays for compounds that modulate CORO1B-dependent functions

• Mechanotransduction research:

  • Investigate how mechanical forces alter CORO1B localization and function

  • Examine the relationship between CORO1B and mechanosensitive junction proteins

  • Study how shear stress affects CORO1B-dependent cytoskeletal remodeling

These applications would benefit from combining CORO1B antibody-based detection with functional assays of endothelial cell behavior in both in vitro and in vivo models of vascular disease.

What novel techniques could enhance CORO1B antibody applications in cytoskeletal research?

Emerging technologies offer opportunities to extend the utility of CORO1B antibodies in studying cytoskeletal dynamics:

• Super-resolution microscopy:

  • Apply techniques like STORM, PALM, or STED to resolve CORO1B localization relative to actin structures at nanoscale resolution

  • Combine with proximity ligation assays to visualize protein-protein interactions in situ

  • Implement expansion microscopy to physically enlarge specimens for enhanced resolution with standard confocal microscopy

• Intravital imaging approaches:

  • Develop protocols for using CORO1B antibodies in tissue clearing techniques (CLARITY, iDISCO)

  • Combine with multiphoton microscopy for deeper tissue imaging

  • Integrate with vascular window models for longitudinal studies in living animals

• Quantitative analysis methods:

  • Implement machine learning algorithms for automated detection of CORO1B-positive structures

  • Develop computational approaches for quantifying dynamic CORO1B-actin associations

  • Apply correlation analysis to map relationships between CORO1B and other junction/cytoskeletal proteins

• Functional genomics integration:

  • Combine CORO1B antibody staining with CRISPR screens targeting cytoskeletal regulators

  • Correlate protein localization patterns with transcriptomic profiles at single-cell resolution

  • Develop antibody-based proximity labeling methods to identify context-specific CORO1B interaction partners

• Microfluidic applications:

  • Design devices to study CORO1B dynamics under controlled flow conditions

  • Develop gradient systems to investigate CORO1B responses to chemotactic stimuli

  • Create organ-on-chip models incorporating CORO1B visualization to study vascular function

These technological advances would provide deeper insights into how CORO1B orchestrates cytoskeletal remodeling in diverse cellular contexts and disease states.