cdc42se1 Antibody

What is CDC42SE1 and why is it significant in cellular research?

CDC42SE1 is a small effector protein of 9 kDa that consists of 79 amino acids, including a conserved CRIB (Cdc42- and Rac-interactive binding) domain, 2 conserved cysteines at the N-terminus, and a basic amino acid preceding the CRIB domain . This protein interacts with CDC42, a small Rho GTPase that plays critical roles in many cellular processes including cell proliferation, survival, cytoskeletal reorganization, and cell motility .

CDC42SE1 is significant in cellular research because:

• It regulates CDC42-induced changes to cell morphology

• It plays a role in actin cytoskeletal remodeling at immunological synapses

• It participates in early contractile events in phagocytosis in macrophages

• It has been shown to inhibit CDC42-induced JNK activity

• Its expression is reduced in skin cancer samples, suggesting a potential tumor suppressor role

What are the standard applications for CDC42SE1 antibodies in laboratory research?

CDC42SE1 antibodies are employed in multiple research applications:

When selecting an antibody, researchers should consider both monoclonal and polyclonal options based on their specific experimental requirements .

How can I validate the specificity of a CDC42SE1 antibody for my research?

Validating CDC42SE1 antibody specificity is crucial for reliable research outcomes. Implement the following validation approaches:

• Positive/negative tissue controls: Use human tonsillitis tissue as a positive control for CDC42SE1 expression . Compare with tissues known to have low expression.

• Overexpression validation: Transfect cells with CDC42SE1 expression vectors and confirm increased antibody signal compared to control cells .

• Knockdown/knockout validation: Verify reduced signal in CDC42SE1 siRNA or CRISPR knockout cells.

• Western blot analysis: Confirm the antibody detects a band of appropriate molecular weight (~9 kDa) .

• Cross-reactivity assessment: Test against recombinant CDC42SE1 protein alongside related proteins (like CDC42SE2) to ensure specificity .

• Peptide competition assay: Pre-incubate the antibody with purified CDC42SE1 protein/peptide to demonstrate signal neutralization.

• Multiple antibodies approach: Compare results using antibodies targeting different epitopes of CDC42SE1 .

What are the key differences between polyclonal and monoclonal CDC42SE1 antibodies?

What are the optimal protocols for detecting CDC42SE1 in tissue samples via immunohistochemistry?

Immunohistochemistry Protocol for CDC42SE1 Detection:

• Sample preparation:

  • Fix tissue in 10% neutral buffered formalin and embed in paraffin

  • Section tissues at 4-5 μm thickness

• Antigen retrieval (critical step):

  • Perform heat-induced epitope retrieval using Tris-EDTA buffer (pH 9.0)

  • Alternative: use citrate buffer (pH 6.0)

  • Heat in pressure cooker or microwave for 15-20 minutes

• Blocking and antibody incubation:

  • Block with 1% BSA for 45 minutes at room temperature

  • Incubate with anti-CDC42SE1 antibody (1:50-1:500 dilution) overnight at 4°C

  • Incubate with appropriate secondary antibody for 1 hour at room temperature

• Detection and visualization:

  • For chromogenic detection, develop with DAB substrate

  • For fluorescent detection, use fluorophore-conjugated secondary antibodies

  • Counterstain nuclei with hematoxylin or DAPI

• Controls:

  • Use human tonsillitis tissue as positive control

  • Include a negative control by omitting primary antibody

Optimization may be necessary for specific tissue types, as CDC42SE1 expression varies by tissue, with higher expression reported in T-lymphocytes, dendritic cells, and whole blood cells .

How can I optimize Western blot protocols for CDC42SE1 detection?

Optimized Western Blot Protocol for CDC42SE1:

• Sample preparation:

  • Extract proteins using RIPA buffer with protease inhibitors

  • Include phosphatase inhibitors if studying phosphorylation states

  • Load 20-40 μg of total protein per lane

• Gel electrophoresis considerations:

  • Use 15-20% SDS-PAGE gels due to CDC42SE1's small size (8.925 kDa)

  • Alternative: Use 4-20% gradient gels for optimal resolution

  • Include positive control (A431 cells or transfected cells overexpressing CDC42SE1)

• Transfer parameters:

  • Use PVDF membrane (0.2 μm pore size) for small proteins

  • Transfer at low voltage (25-30V) overnight at 4°C for small proteins

  • Verify transfer with reversible protein stain

• Antibody incubation:

  • Block with 5% non-fat milk or BSA in TBST

  • Incubate with CDC42SE1 antibody at 1:500-1:1000 dilution

  • Use gentle agitation overnight at 4°C

  • Wash thoroughly with TBST (4-5 times, 5 minutes each)

• Detection optimization:

  • Use high-sensitivity ECL substrate due to potentially low expression levels

  • Consider longer exposure times (2-15 minutes)

  • For weak signals, consider signal enhancers or amplification systems

• Troubleshooting tips:

  • If no band is detected, check loading control first

  • CDC42SE1 may run slightly differently than predicted (8-10 kDa range)

  • Consider enrichment steps (e.g., immunoprecipitation) for low abundance

What experimental approaches can I use to investigate CDC42SE1's interaction with CDC42?

Multiple complementary techniques can robustly analyze CDC42SE1-CDC42 interactions:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate CDC42 using anti-CDC42 antibodies and probe for CDC42SE1

  • Perform reciprocal Co-IP using CDC42SE1 antibodies

  • Include appropriate controls: IgG control, CDC42SE1 H38A mutant (defective in CRIB domain)

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ with high sensitivity

  • Use antibodies against CDC42SE1 and CDC42 from different host species

  • Visualize interaction as fluorescent dots under microscope

• Fluorescence resonance energy transfer (FRET):

  • Create fusion constructs: CDC42SE1-CFP and CDC42-YFP

  • Measure energy transfer between fluorophores when proteins interact

  • Use CDC42SE1 H38A mutant as negative control

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein fused to CDC42SE1 and CDC42

  • Reconstitution of fluorescence when proteins interact

  • Allows visualization of interaction in live cells

• Pull-down assays with recombinant proteins:

  • Express GST-CDC42SE1 and His-tagged CDC42 (GTP-bound form)

  • Perform pull-down and analyze by immunoblotting

  • Compare wild-type CDC42SE1 with H38A mutant

• Surface Plasmon Resonance (SPR):

  • Determine binding kinetics and affinity constants

  • Immobilize purified CDC42-GTP or CDC42SE1 on sensor chip

  • Measure real-time binding parameters

How is CDC42SE1 expression altered in cancer, and what methodologies can detect these changes?

Research has identified significant alterations in CDC42SE1 expression in cancer, particularly skin cancer:

• Expression patterns in cancer:

  • CDC42SE1 is downregulated in human squamous cell carcinoma (SCC) compared to matched perilesional controls

  • Reduced expression promotes tumorigenesis, suggesting a tumor suppressor role

  • CDC42SE1 might be an important marker of skin cancer progression

• Quantitative detection methods:

  • qRT-PCR: For quantifying mRNA expression levels

    • Primers: 5′-CGGATTGACCGGACCATGATT and 5′-CGGTTTCCCTTGGATCTCATCT

    • Normalize with housekeeping genes like MRPL-27

  • Western blot analysis: For protein expression quantification

    • Use densitometry to compare expression levels

    • Normalize to loading controls (β-actin, GAPDH)

  • Immunohistochemistry: For tissue expression patterns

    • Score staining intensity (0-3+) and percentage of positive cells

    • Apply H-score or Allred scoring systems for quantification

    • Compare tumor samples with matched normal tissues

• Functional validation approaches:

  • Overexpression of CDC42SE1 in cancer cell lines (e.g., A431 cells) reduces:

    • Cell proliferation

    • Colony formation in soft agar

    • Tumor volume in nude mice xenografts

  • Mechanistic investigations show:

    • CDC42SE1 inhibits the Akt pathway

    • Reduces phosphorylated Akt (p-Akt) and mTOR (p-mTOR) levels

    • Requires intact CRIB domain for CDC42 binding and tumor suppressive effects

What techniques can be used to study the role of CDC42SE1 in actin cytoskeleton dynamics?

CDC42SE1 plays important roles in actin cytoskeleton remodeling. The following techniques are valuable for investigating these functions:

• Fluorescence microscopy of F-actin structures:

  • Stain F-actin with fluorescently-labeled phalloidin

  • Co-stain with CDC42SE1 antibodies to assess colocalization

  • Quantify actin structures (filopodia, stress fibers) in CDC42SE1-manipulated cells

• Live cell imaging with fluorescent fusion proteins:

  • Generate CDC42SE1-GFP fusion constructs

  • Use LifeAct-RFP to visualize F-actin dynamics

  • Perform time-lapse microscopy to track dynamic changes

• Immunological synapse formation assays:

  • Study CDC42SE1's role in F-actin accumulation at immunological synapses

  • Co-culture T cells with antigen-presenting cells

  • Visualize and quantify F-actin accumulation at the interface

• Phagocytosis assays:

  • Assess CDC42SE1's role in contractile events during phagocytosis

  • Use fluorescently-labeled particles or bacteria

  • Measure phagocytic efficiency in cells with modified CDC42SE1 expression

• Actin polymerization assays:

  • Measure the rate of actin polymerization in vitro

  • Use pyrene-labeled actin to monitor polymerization kinetics

  • Add purified CDC42SE1 and CDC42 proteins to the reaction

• Super-resolution microscopy:

  • Apply techniques like STORM or PALM for nanoscale imaging

  • Resolve detailed actin structures beyond diffraction limit

  • Precisely localize CDC42SE1 in relation to actin filaments

How can I use CDC42SE1 antibodies to investigate its role in cell signaling pathways?

CDC42SE1 influences several signaling pathways, particularly through its interaction with CDC42. Here are methodological approaches to investigate these pathways:

• Phosphorylation state analysis:

  • Western blot with phospho-specific antibodies for key signaling proteins:

    • Akt (p-Akt at Ser473 and Thr308)

    • mTOR (p-mTOR at Ser2448)

    • PAK1 (p-PAK1 at Thr423)

  • Compare signaling dynamics in cells with normal, overexpressed, or knocked-down CDC42SE1

• Pathway inhibitor studies:

  • Use specific inhibitors (e.g., PI3K inhibitors, mTOR inhibitors)

  • Determine if CDC42SE1's effects are dependent on specific pathways

  • Assess if inhibitors can reverse phenotypes caused by CDC42SE1 manipulation

• Antibody microarray analysis:

  • Apply cell lysates to antibody microarrays detecting multiple signaling proteins

  • Compare signaling profiles between control and CDC42SE1-manipulated cells

  • Use pathway analysis software (e.g., Ingenuity Pathway Analysis) to identify affected networks

• Reporter assays:

  • Utilize luciferase reporters for pathways potentially affected by CDC42SE1

  • Test JNK pathway reporters based on CDC42SE1's known inhibitory effect on CDC42-induced JNK activity

  • Measure transcriptional outcomes of pathway activation/inhibition

• Phosphoproteomics:

  • Perform mass spectrometry-based phosphoproteomics

  • Identify global changes in phosphorylation patterns

  • Discover novel signaling events regulated by CDC42SE1

What are the best practices for troubleshooting inconsistent results with CDC42SE1 antibodies?

When encountering inconsistent results with CDC42SE1 antibodies, systematically address these common issues:

• Antibody validation issues:

  • Verify antibody specificity using positive and negative controls

  • Test multiple antibodies targeting different epitopes of CDC42SE1

  • Consider lot-to-lot variability, especially with polyclonal antibodies

• Sample preparation challenges:

  • CDC42SE1 is a small protein (8.925 kDa) that may require specific extraction methods

  • Ensure complete protein extraction with appropriate lysis buffers

  • Consider subcellular fractionation as CDC42SE1 localizes to both cytoplasm/cytoskeleton and membrane

• Technical optimization strategies:

  • For Western blot:

    • Use high percentage gels (15-20%) for better resolution of small proteins

    • Optimize transfer conditions for small proteins (longer times, lower voltage)

    • Try different blocking reagents (milk vs. BSA) if background is an issue

  • For IHC/ICC:

    • Test multiple antigen retrieval methods (pH 6.0 vs. pH 9.0)

    • Optimize primary antibody concentration (try 1:50-1:500 dilution range)

    • Extend incubation times at lower temperatures (overnight at 4°C)

• Cell/tissue-specific considerations:

  • CDC42SE1 expression varies by tissue type

  • Higher expression in T-lymphocytes, dendritic cells, and whole blood cells

  • Consider using positive control tissues (e.g., human tonsillitis tissue)

• Systematic troubleshooting approach:

  • Change one variable at a time

  • Document all conditions thoroughly

  • Include appropriate positive and negative controls in each experiment