CDC42EP2 Antibody

What is CDC42EP2 and what is its significance in cellular function?

CDC42EP2 (CDC42 Effector Protein 2), also known as BORG1 or CEP2, is a 210 amino acid intracytoplasmic protein that belongs to the Borg family of CDC42 effector proteins. It contains a CRIB (Cdc42/Rac interactive-binding) domain that mediates interaction with CDC42, a small Rho GTPase . CDC42EP2 is primarily involved in:

• Organization of the actin cytoskeleton

• Regulation of cell shape through actin filament assembly

• Induction of pseudopodia formation in fibroblasts

• Potential roles in cell migration and invasion

CDC42EP2 is highly expressed in heart tissue with lower expression levels in liver and pancreas . Recent research has identified CDC42EP2 as a novel prognostic biomarker in liver hepatocellular carcinoma (LIHC), where upregulated expression correlates with worse prognosis .

What applications are CDC42EP2 antibodies typically used for?

CDC42EP2 antibodies are validated for multiple research applications including:

These applications allow researchers to investigate CDC42EP2 expression, localization, and interactions in various experimental contexts .

What species reactivity should be considered when selecting a CDC42EP2 antibody?

When selecting a CDC42EP2 antibody, consider the following species reactivity profiles:

• Most commercially available CDC42EP2 antibodies show strong reactivity with human samples

• Many antibodies also cross-react with mouse CDC42EP2

• Some antibodies demonstrate additional reactivity with rat samples

• Limited antibodies may cross-react with monkey samples

The specific epitope targeted can affect cross-reactivity. For example, antibodies targeting conserved regions (such as the CRIB domain) may show broader species reactivity than those targeting more variable regions . Always validate the antibody for your specific experimental model and species of interest.

How can I optimize Western blot protocols for CDC42EP2 detection?

Optimizing Western blot protocols for CDC42EP2 detection requires attention to several critical parameters:

• Protein Extraction:

  • For cytoskeletal-associated CDC42EP2, include cytoskeleton preservation buffers containing protease inhibitors

  • CDC42EP2 localizes to both cytoplasm and cytoskeleton, so appropriate fractionation may be required for specific studies

• Sample Preparation:

  • CDC42EP2 has a molecular weight of approximately 22-23 kDa

  • Use 4-20% gradient gels for optimal resolution

  • Load 20-40 μg of total protein per lane

• Antibody Concentration:

  • Primary antibody: Use at 1:500-1:1000 dilution (typically 0.2-1 μg/mL)

  • Secondary antibody: Anti-rabbit HRP at 1:5000-1:10000

• Controls:

  • Positive controls: K-562 cells and mouse pancreas tissue show consistent expression

  • Blocking peptide: Use the immunogen peptide to confirm specificity

• Detection System:

  • Enhanced chemiluminescence (ECL) is suitable due to the moderate expression levels of CDC42EP2

  • Consider longer exposure times (1-5 minutes) for detecting endogenous levels

The purification method of the antibody (protein A/G purification versus antigen affinity purification) can significantly impact sensitivity and specificity .

What are the most effective protocols for investigating CDC42EP2 interactions with CDC42 and the actin cytoskeleton?

For investigating CDC42EP2 interactions with CDC42 and the actin cytoskeleton, researchers should consider these methodological approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-CDC42EP2 antibody for pull-down experiments

  • Crosslinking may be required to capture transient interactions

  • Analyze precipitates for CDC42 and other cytoskeletal components

• Fluorescence Co-localization:

  • Implement double immunofluorescence with CDC42EP2 antibody and phalloidin (for F-actin)

  • Use super-resolution microscopy for detailed co-localization analysis

  • Quantify Pearson's correlation coefficients between CDC42EP2 and cytoskeletal markers

• Proximity Ligation Assay (PLA):

  • Enables visualization of protein-protein interactions within 40 nm

  • Combine CDC42EP2 antibody with anti-CDC42 or anti-actin antibodies

  • Provides higher specificity than conventional co-localization

• FRET Analysis:

  • For live cell imaging of CDC42EP2-CDC42 interactions

  • Requires fluorescently tagged proteins but provides dynamic interaction data

• Functional Analysis:

  • Compare actin organization in CDC42EP2-depleted versus control cells

  • Assess pseudopodia formation in fibroblasts as a readout of CDC42EP2 activity

Research indicates that CDC42EP5 (another BORG family member) promotes actomyosin function through SEPT9 actin cross-linking . Similar methodologies could be applied to investigate CDC42EP2's potential role in these processes.

What technical considerations are important when using CDC42EP2 antibodies for immunohistochemistry?

When using CDC42EP2 antibodies for immunohistochemistry, consider these technical aspects:

• Tissue Preparation:

  • Formalin-fixed paraffin-embedded (FFPE) tissues have been validated for CDC42EP2 detection

  • Citrate buffer (pH 6.0) is recommended for antigen retrieval

  • Optimal section thickness: 4-6 μm

• Antibody Selection and Dilution:

  • For FFPE tissues: Use antibodies at 1:20-1:200 dilution

  • Polyclonal antibodies typically provide stronger signal in IHC applications

  • Antibodies targeting C-terminal regions may yield better results in fixed tissues

• Visualization System:

  • DAB (3,3'-diaminobenzidine) detection systems are commonly used

  • Signal amplification may be required for low-expressing tissues

  • Hematoxylin counterstaining provides context for cellular localization

• Controls and Validation:

  • Positive tissue controls: Human heart tissue, gastric cancer

  • Peptide competition assay to confirm specificity

  • CDC42EP2 shows cytoplasmic and membrane localization patterns

• Quantification Approaches:

  • H-score system for semi-quantitative analysis

  • Digital pathology solutions for precise quantification

  • Consider both intensity and percentage of positive cells

As shown in research studies, CDC42EP2 expression in tissues can have prognostic value, particularly in liver hepatocellular carcinoma .

How can CDC42EP2 antibodies be effectively used in cancer research?

CDC42EP2 antibodies offer valuable tools for cancer research applications:

• Prognostic Biomarker Analysis:

  • Recent studies show CDC42EP2 is upregulated in liver hepatocellular carcinoma (LIHC)

  • Higher expression correlates with worse prognosis

  • IHC with CDC42EP2 antibodies can evaluate expression in patient tissue samples

• Tumor-Infiltrating Immune Cell Studies:

  • CDC42EP2 shows positive association with tumor-infiltrating immune cells (TIICs)

  • Combined staining with immune cell markers and CDC42EP2 antibodies can elucidate these relationships

  • May help identify patients who could benefit from immunotherapy

• Cell Migration and Invasion Assays:

  • CDC42EP2 may regulate cell motility similar to CDC42EP5

  • Antibody staining can track changes in CDC42EP2 localization during cell migration

  • Functional blocking studies using antibodies can help determine mechanism

• Pathway Analysis:

  • Co-expression analyses show CDC42EP2 is associated with protein localization to endoplasmic reticulum, translational initiation, and RNA catabolic processes

  • CDC42EP2 antibodies can help validate pathway connections identified in omics studies

What are the key considerations when validating CDC42EP2 antibody specificity?

Thorough validation of CDC42EP2 antibody specificity requires multiple complementary approaches:

• Genetic Controls:

  • Use CDC42EP2 knockout/knockdown cells as negative controls

  • Compare staining patterns in wildtype versus CDC42EP2-depleted samples

  • Overexpression systems can serve as positive controls

• Peptide Competition:

  • Pre-incubate antibody with immunizing peptide

  • Should abolish specific signal in all applications

  • Different concentrations of blocking peptide can determine affinity

• Multiple Antibody Validation:

  • Compare results using antibodies targeting different epitopes of CDC42EP2

  • Antibodies against N-terminal vs. C-terminal regions should show similar patterns

  • Monoclonal and polyclonal antibodies may provide complementary evidence

• Multiple Detection Methods:

  • Combine Western blot, IHC, and IF results

  • Molecular weight confirmation (22-23 kDa) in Western blot

  • Expected cellular localization (cytoplasm and cytoskeleton) in microscopy

• Mass Spectrometry Confirmation:

  • Immunoprecipitate with CDC42EP2 antibody

  • Verify pulled-down protein identity by mass spectrometry

  • Can identify potential cross-reacting proteins

Antibody specificity testing is crucial as the BORG/CEP family contains multiple members (CDC42EP1-5) with structural similarities that could lead to cross-reactivity .

How can CDC42EP2 antibodies be used to investigate B cell development and immune response mechanisms?

CDC42EP2 antibodies can provide valuable insights into B cell development and immune responses, though this application builds on broader CDC42 pathway research:

• B Cell Development Analysis:

  • While CDC42EP2-specific roles in B cells are not fully characterized, the CDC42 pathway is crucial for B cell development

  • CDC42 deletion causes severe defects in B cell development, plasma cell differentiation, and antigen presentation

  • CDC42EP2 antibodies can help determine if these effects are mediated through CDC42EP2

• Germinal Center Formation:

  • CDC42 is required for germinal center formation during infection

  • CDC42EP2 antibodies can assess its expression and localization in germinal centers

  • Potential correlation with CDC42 activation status in these structures

• Antibody Response Investigation:

  • CDC42 is critical for mounting antibody responses to T cell-dependent antigens

  • CDC42EP2 antibodies can help determine if CDC42EP2 is involved in this process

  • Combined with flow cytometry markers for plasma cells and memory B cells

• Cytoskeletal Dynamics in Immune Cells:

  • CDC42EP2 regulates actin cytoskeleton organization

  • Antibodies can visualize its distribution during immune synapse formation

  • May reveal roles in immune cell migration and interaction

• B-T Cell Interaction Studies:

  • CDC42 deficiency impairs B-T cell interactions in vivo

  • CDC42EP2 antibodies can assess its localization during these cellular interactions

  • Potential involvement in immunological synapse formation

While direct evidence for CDC42EP2's role in B cells is still emerging, its position in the CDC42 pathway—which has profound effects on B cell function—suggests it may contribute to these processes .

What strategies can address weak or absent CDC42EP2 signal in Western blot applications?

When encountering weak or absent CDC42EP2 signal in Western blots, consider these troubleshooting strategies:

• Sample Preparation Issues:

  • CDC42EP2 is cytoskeletal-associated; use appropriate lysis buffers with cytoskeleton preservation components

  • Avoid excessive freeze-thaw cycles that may degrade the protein

  • Include protease inhibitors to prevent degradation

  • For cell fractionation, check both cytoplasmic and cytoskeletal fractions

• Detection Sensitivity:

  • Increase protein loading (40-60 μg)

  • Reduce antibody dilution (1:250-1:500)

  • Use enhanced chemiluminescence (ECL) substrates with higher sensitivity

  • Consider signal amplification systems or longer exposure times

• Antibody Selection:

  • CDC42EP2 has multiple epitopes; try antibodies targeting different regions

  • C-terminal antibodies (e.g., AA 133-160) may provide better detection

  • Switch between monoclonal and polyclonal antibodies

• Transfer Optimization:

  • For low molecular weight CDC42EP2 (~22 kDa):

    • Use 0.2 μm PVDF membranes instead of 0.45 μm

    • Reduce transfer time or voltage to prevent protein pass-through

    • Consider semi-dry transfer for small proteins

• Positive Controls:

  • Include validated positive controls like K-562 cells or mouse pancreas tissue

  • Consider loading recombinant CDC42EP2 protein as reference

  • Test multiple cell lines as CDC42EP2 expression varies between tissues

Technical data shows that observed molecular weight is approximately 22-23 kDa, and antibody dilutions between 1:500-1:1000 are typically effective for detection .

How can researchers address potential cross-reactivity with other CDC42 effector proteins?

Addressing potential cross-reactivity with other CDC42 effector proteins requires strategic approaches:

• Epitope Selection:

  • Choose antibodies targeting unique regions of CDC42EP2

  • Avoid antibodies targeting the highly conserved CRIB domain shared among CDC42 effector proteins

  • C-terminal regions tend to have greater sequence divergence among BORG family members

• Validation in Knockout/Knockdown Systems:

  • Generate CDC42EP2-specific knockdowns or knockouts

  • Test antibody reactivity in these systems

  • Signal should be absent or significantly reduced

• Multiple Antibody Approach:

  • Use antibodies from different sources targeting different epitopes

  • Compare staining patterns and signal intensities

  • Consistent results across antibodies increase confidence in specificity

• Immunoprecipitation-Mass Spectrometry:

  • Perform IP with CDC42EP2 antibody followed by mass spectrometry

  • Identify all pulled-down proteins

  • Assess presence of other CDC42 effector proteins in the precipitate

• Western Blot Analysis:

  • Look for multiple bands that might indicate cross-reactivity

  • Compare observed molecular weights with predicted weights of other BORG family members:

    • CDC42EP1: ~38 kDa

    • CDC42EP2: ~22 kDa

    • CDC42EP3: ~23 kDa

    • CDC42EP4: ~38 kDa

    • CDC42EP5: ~30 kDa

Research indicates that CDC42EP5 has distinct functions from CDC42EP2 in certain contexts, highlighting the importance of antibody specificity when studying individual family members .

What are the most common technical challenges in immunohistochemical detection of CDC42EP2 and how can they be addressed?

Researchers commonly encounter these technical challenges when performing immunohistochemical detection of CDC42EP2:

• Fixation and Antigen Retrieval Issues:

  • CDC42EP2 epitopes may be masked by formalin fixation

  • Solution: Optimize antigen retrieval methods

    • Try heat-induced epitope retrieval with citrate buffer (pH 6.0)

    • Compare with EDTA buffer (pH 9.0)

    • Adjust retrieval time (15-30 minutes)

• Background Signal:

  • Non-specific binding can obscure true CDC42EP2 signal

  • Solution: Implement blocking optimization

    • Use protein block containing 2-5% BSA and 5-10% normal serum

    • Consider avidin/biotin block if using biotin-based detection

    • Include 0.1-0.3% Triton X-100 to reduce cytoplasmic background

• Signal Intensity Variation:

  • CDC42EP2 expression levels vary between tissues

  • Solution: Adjust antibody concentration by tissue type

    • For high-expressing tissues (heart): Use 1:100-1:200 dilution

    • For low-expressing tissues: Use 1:20-1:50 dilution

    • Consider signal amplification systems for low-expressing tissues

• Cell-Type Specificity:

  • Determining which cells express CDC42EP2 in heterogeneous tissues

  • Solution: Implement dual staining approaches

    • Combined staining with cell-type markers

    • Sequential immunohistochemistry with digital image overlay

    • Multiplex immunofluorescence for co-localization studies

• Quantification Challenges:

  • CDC42EP2 shows both cytoplasmic and membrane localization

  • Solution: Adopt appropriate scoring systems

    • Use H-score system for combined intensity/percentage assessment

    • Digital pathology with cellular compartment analysis

    • Develop tissue-specific scoring thresholds based on clinical correlations

Published research has successfully detected CDC42EP2 in paraffin-embedded human gastric cancer and heart tissue using antibody dilutions of 1:100, demonstrating these approaches can be effective .

How might CDC42EP2 antibodies be utilized to explore its potential role as a therapeutic target in cancer?

CDC42EP2 antibodies can facilitate exploration of its therapeutic potential in cancer through several research avenues:

• Expression Profiling Across Cancer Types:

  • Use CDC42EP2 antibodies for tissue microarray analysis

  • Compare expression between tumor and normal tissues across cancer types

  • Correlate expression levels with clinical outcomes to identify cancer types where CDC42EP2 targeting would be most beneficial

• Functional Inhibition Studies:

  • Develop function-blocking antibodies targeting CDC42EP2

  • Assess effects on cancer cell proliferation, migration, and invasion

  • Compare with genetic knockdown approaches to validate specificity

• Mechanism Exploration:

  • Investigate CDC42EP2 interaction partners using antibody-based techniques:

    • Co-immunoprecipitation followed by mass spectrometry

    • Proximity ligation assays to validate interactions in situ

    • Identify druggable nodes in CDC42EP2-dependent pathways

• Tumor Microenvironment Analysis:

  • CDC42EP2 shows association with tumor-infiltrating immune cells

  • Use multicolor immunofluorescence with CDC42EP2 antibodies and immune cell markers

  • Identify potential immunomodulatory effects of CDC42EP2 inhibition

• Biomarker Development:

  • Standardize CDC42EP2 IHC protocols for clinical application

  • Develop scoring systems correlating with treatment response

  • Evaluate CDC42EP2 as a companion diagnostic for targeted therapies

Research indicates CDC42EP2 is a novel prognostic biomarker in liver hepatocellular carcinoma, with upregulated expression associated with worse prognosis, suggesting its potential as a therapeutic target .

What is the current understanding of CDC42EP2's role in cytoskeletal dynamics, and how can antibodies help advance this knowledge?

Current understanding of CDC42EP2's role in cytoskeletal dynamics can be expanded using antibodies in these research directions:

• Localization During Dynamic Cellular Processes:

  • CDC42EP2 is involved in actin cytoskeleton organization

  • Antibodies can track its distribution during:

    • Cell migration and invasion

    • Cell division

    • Response to mechanical stimuli

  • Time-lapse imaging with live-cell compatible antibody formats

• Interaction with Cytoskeletal Regulators:

  • CDC42EP2 acts downstream of CDC42 to induce actin filament assembly

  • Antibodies can help map interactions with:

    • Actin nucleation factors

    • Myosin regulatory proteins

    • Septin family members (potential similarity to CDC42EP5-SEPT9 interaction)

  • Co-immunoprecipitation and proximity ligation assays are key methodologies

• Role in Specialized Cellular Structures:

  • CDC42EP2 induces pseudopodia formation in fibroblasts

  • Antibodies can assess its presence in:

    • Lamellipodia and filopodia

    • Invadopodia in cancer cells

    • Cell-cell junctions

  • Super-resolution microscopy with CDC42EP2 antibodies enables detailed localization

• Post-Translational Modification Analysis:

  • Potential regulation through phosphorylation or other modifications

  • Combination of phospho-specific antibodies with CDC42EP2 antibodies

  • Analysis of modification patterns during cytoskeletal remodeling

• Comparative Analysis with Other BORG Family Members:

  • CDC42EP5 has established roles in actomyosin regulation

  • Antibodies against multiple BORG proteins can determine:

    • Unique vs. redundant functions

    • Cell type-specific roles

    • Potential compensation mechanisms

This area offers significant research potential, as detailed mechanistic understanding of CDC42EP2's cytoskeletal functions remains incomplete despite clear evidence of its involvement in actin organization and cell shape regulation .

How could CDC42EP2 antibodies contribute to understanding its potential role in immune regulation and response to immunotherapy?

CDC42EP2 antibodies could substantially advance understanding of its role in immune regulation and immunotherapy response through these research approaches:

• Immune Cell Expression Profiling:

  • CDC42EP2 has positive associations with tumor-infiltrating immune cells (TIICs)

  • Antibodies can map expression across immune cell subsets:

    • T cell populations (CD4+, CD8+, Tregs)

    • B cell lineages

    • Myeloid cells (macrophages, dendritic cells)

  • Flow cytometry and immunohistochemistry with immune cell markers

• Tumor-Immune Interaction Studies:

  • CDC42 plays critical roles in B-T cell interactions

  • CDC42EP2 antibodies can assess its involvement in:

    • Immunological synapse formation

    • Antigen presentation processes

    • Immune cell migration within tumor microenvironment

  • Multiplex immunofluorescence in tumor tissues

• Response to Immunotherapy Biomarker Development:

  • Correlate CDC42EP2 expression with response to immune checkpoint inhibitors

  • Prospective studies using standardized IHC protocols

  • Potential development as companion diagnostic for immunotherapy

• Functional Impact on Immune Signaling:

  • CDC42EP2 may regulate cytoskeletal dynamics in immune cells similar to its role in other cell types

  • Antibodies can map its localization during:

    • T cell receptor signaling

    • B cell receptor engagement

    • Phagocytosis in myeloid cells

  • Combined with phospho-flow or calcium flux assays

• In vivo Immune Response Models:

  • Use CDC42EP2 antibodies to track expression changes during:

    • Vaccination responses

    • Infectious disease models

    • Autoimmune conditions

  • Potential correlations with antibody production and cell-mediated immunity