G2E3 Antibody

What is G2E3 and why is it important in cellular research?

G2E3 (G2/M-phase specific E3 ubiquitin protein ligase) is a nucleo-cytoplasmic shuttling protein with a C-terminal HECT domain that functions in protein ubiquitination . It plays a significant role in DNA damage response (DDR) pathways and the regulation of apoptosis . The canonical human G2E3 protein has 706 amino acid residues with a molecular mass of approximately 80.5 kDa . G2E3 is particularly important in research because it has been identified as a potential target in cancer therapy, as its depletion sensitizes cancer cells to DNA-damaging agents . G2E3 is highly expressed in the brain, liver, kidney, testes, and ovary, suggesting tissue-specific functions that warrant investigation .

What are the key characteristics of G2E3 antibodies?

G2E3 antibodies are immunological reagents designed for the detection and study of G2E3 protein. These antibodies can be used in various applications including Western blotting and ELISA . When selecting a G2E3 antibody, researchers should consider specificity (ability to distinguish G2E3 from other ubiquitin ligases), sensitivity (detection threshold), and compatibility with intended applications. G2E3 antibodies may target different epitopes within the protein structure, which can affect their suitability for specific experimental conditions and techniques . Due to the nuclear-cytoplasmic shuttling nature of G2E3, antibodies capable of detecting both nuclear and cytoplasmic fractions are particularly valuable for comprehensive studies .

How does G2E3 function in the DNA damage response pathway?

G2E3 functions as a modulator of the DNA damage response (DDR). Research has shown that G2E3 depletion decreases the phosphorylation of H2AX (γH2AX) and checkpoint kinase 1 in response to cisplatin treatment . This suggests that G2E3 plays a role in signal transduction following DNA damage. Additionally, G2E3 acts as a negative regulator of p53 activity, as its knockdown leads to increased p53 and p21 levels . The protein appears to be involved in maintaining genomic stability during DNA replication, as G2E3 depletion increases the accumulation of single-stranded DNA upon gemcitabine treatment, indicating heightened replicative stress . Interestingly, G2E3 itself is downregulated in response to various DNA-damaging agents including cisplatin, gemcitabine, and neocarzinostatin, suggesting a feedback regulatory mechanism in the DDR pathway .

What are the most effective applications for detecting G2E3 in cellular samples?

Based on research findings, several methods have proven effective for G2E3 detection:

• Immunoblotting/Western Blot: Western blotting has been widely used to detect G2E3 protein levels, though some studies note that commercial antibodies may not directly detect endogenous G2E3 in standard immunoblots of cell lysates, requiring immunoprecipitation-immunoblot protocols for quantification .

• Immunofluorescence: This technique has been successfully employed to visualize the subcellular localization of G2E3 and to assess changes in protein levels upon treatment with DNA-damaging agents .

• Quantitative RT-PCR: For analyzing G2E3 mRNA expression levels, qRT-PCR has been reliably used in multiple studies, particularly when examining changes in G2E3 expression following treatments .

• Immunoprecipitation: Due to potential detection challenges, immunoprecipitation followed by immunoblotting has been utilized to concentrate and detect G2E3 protein .

• ELISA: This method has also been documented as an effective application for G2E3 antibodies .

The choice of method should be determined by the specific research question, available resources, and whether protein localization, expression levels, or protein-protein interactions are being investigated.

How should researchers validate the specificity of G2E3 antibodies?

Validating G2E3 antibody specificity is crucial for reliable research outcomes. A comprehensive validation approach should include:

• Knockdown/Knockout Controls: Perform siRNA-mediated knockdown of G2E3 (using multiple siRNAs targeting different regions as demonstrated in previous research) and compare antibody signal between control and knockdown samples . The significant reduction in signal in knockdown samples confirms specificity.

• Overexpression Controls: Express tagged G2E3 constructs (e.g., HA-tagged or GFP-tagged G2E3 as used in published studies) and confirm co-detection with the G2E3 antibody .

• Molecular Weight Verification: Ensure that the detected band corresponds to the expected molecular weight of G2E3 (approximately 80.5 kDa for the canonical human protein) .

• Cross-Reactivity Testing: If working with multiple species, test the antibody against samples from different organisms to confirm cross-reactivity with orthologs, as G2E3 orthologs have been reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken .

• Epitope Competition Assay: When possible, perform competition assays with purified G2E3 protein or specific peptides to confirm binding specificity .

What protein extraction methods are most suitable for G2E3 detection by Western blot?

For optimal G2E3 detection via Western blot, researchers should consider the following extraction methods:

• Standard Mammalian Cell Lysis Buffer (MCLB): This has been effectively used in published studies for G2E3 detection . The protocol typically involves:

  • Harvesting cells and lysing them in MCLB

  • Clearing lysates by centrifugation

  • Quantifying proteins using Bradford assay

  • Separating proteins on SDS-polyacrylamide gels

• Nuclear and Cytoplasmic Fractionation: Since G2E3 is a nucleo-cytoplasmic shuttling protein, separate extraction of nuclear and cytoplasmic fractions may be beneficial for studying its subcellular distribution and regulation .

• Immunoprecipitation Protocol: For cases where direct detection is challenging, immunoprecipitation followed by immunoblotting has been shown to effectively quantify G2E3 levels .

• Protein Denaturation Conditions: Ensure complete protein denaturation through appropriate buffer composition and heating conditions to expose the epitope for antibody recognition.

• Protease and Phosphatase Inhibitors: Include these in extraction buffers, particularly when studying post-translational modifications or when working with samples treated with DNA-damaging agents that may affect G2E3 stability .

After extraction, proteins should be transferred to nitrocellulose membranes for immunoblotting according to standard protocols .

How can G2E3 antibodies be utilized to study DNA damage response pathways?

G2E3 antibodies can be powerful tools for investigating DNA damage response (DDR) pathways through several advanced approaches:

• Chromatin Immunoprecipitation (ChIP): Use G2E3 antibodies to identify genomic regions where G2E3 may associate with chromatin in response to DNA damage, potentially revealing its direct involvement in DNA repair processes.

• Proximity Ligation Assays: Combine G2E3 antibodies with antibodies against known DDR proteins (such as H2AX, Chk1, or p53) to visualize and quantify protein-protein interactions in situ following DNA damage .

• Immunofluorescence Co-localization: Study the spatial and temporal dynamics of G2E3 in relation to DDR proteins like γH2AX following treatment with DNA-damaging agents such as cisplatin or gemcitabine .

• Phosphorylation-Specific Detection: Develop or utilize antibodies that specifically recognize post-translationally modified forms of G2E3 that may arise during the DDR.

• Quantitative Analysis of DDR Signaling: Monitor changes in G2E3 levels in relation to other DDR components like H2AX phosphorylation, p53 accumulation, and caspase activation to establish signaling hierarchies and feedback mechanisms .

Research has demonstrated that G2E3 depletion affects H2AX phosphorylation and p53 levels, suggesting that tracking these relationships using antibody-based methods can provide insights into G2E3's role in DDR regulation .

What are the challenges in detecting endogenous G2E3 protein and how can they be overcome?

Detecting endogenous G2E3 protein presents several challenges that researchers should be aware of:

• Low Abundance: G2E3 may be expressed at low levels in some cell types or under certain conditions, making detection difficult. This can be addressed by:

  • Using enhanced chemiluminescence detection systems with increased sensitivity

  • Employing signal amplification methods

  • Concentrating the protein through immunoprecipitation before analysis

• Antibody Limitations: Published research notes that "none of the available antibodies to G2E3 detected the protein in blots of cell lysates," necessitating alternative approaches :

  • Implementing immunoprecipitation-immunoblot protocols for endogenous G2E3 detection

  • Using tagged G2E3 constructs for overexpression studies

  • Validating antibody functionality in multiple applications

• Cell Type and Condition Specificity: G2E3 expression varies across tissues and is affected by treatments such as DNA-damaging agents :

  • Select cell lines known to express higher levels of G2E3 (brain, liver, kidney, testes, or ovary-derived cells)

  • Consider the timing of analysis, as G2E3 levels decrease following exposure to DNA-damaging agents

• Protein Stability Issues: Given G2E3's role in ubiquitination and its own regulation by DNA damage:

  • Include proteasome inhibitors in extraction buffers when appropriate

  • Consider the kinetics of G2E3 degradation in experimental design

• Cross-Reactivity Concerns: Ensure antibody specificity through:

  • Using multiple antibodies targeting different epitopes

  • Including appropriate positive and negative controls, particularly G2E3 knockdown samples

How can G2E3 antibodies contribute to understanding cell cycle regulation and apoptosis?

G2E3 antibodies can be instrumental in elucidating the role of G2E3 in cell cycle regulation and apoptosis through several research approaches:

• Cell Cycle Phase-Specific Analysis: As a G2/M-phase specific protein, analyze G2E3 levels and localization across different cell cycle phases using:

  • Synchronized cell populations

  • Co-staining with cell cycle markers

  • Flow cytometry with G2E3 antibodies to correlate expression with cell cycle position

• Apoptosis Pathway Investigation: Research has shown that G2E3 depletion increases susceptibility to apoptosis in both p53-dependent and p53-independent manners :

  • Monitor G2E3 in relation to apoptotic markers (cleaved caspase-3, cleaved PARP-1)

  • Analyze temporal relationships between G2E3 downregulation and initiation of apoptosis

  • Investigate G2E3 interactions with key apoptotic regulators

• Post-Treatment Dynamics: Track changes in G2E3 expression and localization following treatment with:

  • DNA-damaging agents (cisplatin, gemcitabine, neocarzinostatin)

  • Cell cycle inhibitors

  • Apoptosis inducers

• Correlation Analysis: Establish relationships between G2E3 levels and:

  • p53 and p21 expression

  • Caspase activation

  • Cell proliferation rates

  • Replicative stress markers (ssDNA accumulation)

• Pathway Dissection: Use G2E3 antibodies in combination with inhibitors of specific pathways to determine:

  • Whether G2E3 acts upstream or downstream of key regulatory points

  • The epistatic relationships between G2E3 and other regulators (e.g., Mdm2 and p53)

What are common issues when using G2E3 antibodies and how can they be resolved?

Researchers working with G2E3 antibodies may encounter several challenges that require specific troubleshooting approaches:

• Weak or No Signal Detection:

  • Increase protein concentration in samples

  • Optimize antibody concentration and incubation conditions

  • Consider immunoprecipitation-immunoblot protocol as direct detection has been challenging

  • Verify antibody functionality with positive controls (e.g., overexpressed tagged-G2E3)

  • Ensure protein transfer efficiency during Western blotting

• Non-specific Banding:

  • Increase blocking stringency

  • Optimize antibody dilution

  • Perform validation with knockdown controls to identify specific bands

  • Use purified antibody preparations when available

  • Consider pre-adsorption with non-specific proteins

• Inconsistent Results Between Experiments:

  • Standardize cell culture conditions, as G2E3 expression is affected by cell cycle phase and DNA damage

  • Maintain consistent protein extraction and handling protocols

  • Use internal loading controls appropriate for the experimental conditions

  • Consider the timing of analysis, as G2E3 levels change in response to treatments

• Poor Immunofluorescence Staining:

  • Optimize fixation methods (paraformaldehyde has been successfully used)

  • Adjust permeabilization conditions to ensure antibody access to both nuclear and cytoplasmic G2E3

  • Include appropriate controls for autofluorescence and non-specific binding

  • Consider epitope masking issues that may occur during fixation

• Difficulty Detecting Changes in G2E3 Levels:

  • Use quantitative approaches (densitometry for Western blots, quantitative image analysis for immunofluorescence)

  • Consider kinetics of G2E3 regulation when designing experiments

  • Include positive controls known to affect G2E3 levels (e.g., DNA-damaging agents)

How should researchers interpret conflicting results when studying G2E3 across different cell lines?

When confronted with conflicting results across different cell lines, researchers should consider several factors that might explain these variations:

• Cell Type-Specific Expression Patterns:

  • G2E3 is differentially expressed across tissues, with high expression in brain, liver, kidney, testes, and ovary

  • Document baseline G2E3 expression in each cell line via qRT-PCR and protein analysis

  • Consider whether cell origin (tissue type, species, disease state) affects G2E3 function

• p53 Status Considerations:

  • Research has shown both p53-dependent and p53-independent functions of G2E3

  • Determine the p53 status (wild-type, mutant, null) of each cell line

  • Compare results from isogenic cell lines differing only in p53 status (e.g., HCT116 p53+/+ vs. HCT116 p53-/-)

  • Analyze whether observed effects correlate with p53 functionality

• Cell Cycle Distribution Variations:

  • As a G2/M-phase specific protein, G2E3 expression and function may vary with cell cycle distribution

  • Assess cell cycle profiles of different cell lines under experimental conditions

  • Consider synchronizing cells when appropriate for direct comparisons

• Differential DNA Damage Response Pathways:

  • Cell lines may have different intrinsic DDR capacities and mechanisms

  • Compare the kinetics of DDR marker expression (γH2AX, Chk1 phosphorylation) alongside G2E3

  • Evaluate whether differences in G2E3 behavior correlate with other DDR components

• Experimental Design Considerations:

  • Standardize experimental conditions as much as possible across cell lines

  • Use multiple methodologies to confirm observations (e.g., both protein and mRNA analysis)

  • Consider genetic approaches (knockdown, knockout, overexpression) to establish causality rather than correlation

What controls are essential when using G2E3 antibodies in different experimental contexts?

To ensure reliable and interpretable results when using G2E3 antibodies, researchers should implement the following controls based on experimental context:

• Western Blot/Immunoblotting Controls:

  • Positive Control: Lysate from cells known to express G2E3 or cells overexpressing tagged G2E3

  • Negative Control: Lysate from cells with G2E3 knockdown using validated siRNAs

  • Loading Control: Appropriate housekeeping protein (e.g., actin) to normalize protein levels

  • Molecular Weight Marker: To confirm the expected size of G2E3 (approximately 80.5 kDa)

  • Treatment Control: Samples from cells treated with agents known to affect G2E3 levels (e.g., cisplatin)

• Immunofluorescence Controls:

  • Antibody Specificity Control: Cells with G2E3 knockdown to demonstrate specificity

  • Primary Antibody Omission: To assess secondary antibody non-specific binding

  • Nuclear Stain: DAPI or similar to correlate G2E3 localization with nuclear compartment

  • Co-localization Standards: Known nuclear and cytoplasmic markers when studying G2E3 shuttling

• Functional Studies Controls:

  • Multiple siRNAs: Use of different siRNAs targeting G2E3 to rule out off-target effects

  • Rescue Experiment: Re-expression of siRNA-resistant G2E3 to confirm specificity of observed phenotypes

  • Pathway Controls: Include conditions that modulate known G2E3-related pathways (e.g., p53 inhibition/activation)

  • Time Course Controls: Samples collected at multiple time points to capture dynamic changes in G2E3 levels

• Specialized Application Controls:

  • ChIP Controls: Input sample, IgG control, and positive control for a known G2E3-associated region

  • Immunoprecipitation Controls: IgG control, input sample, and known G2E3-interacting protein control

  • Heterokaryon Assay: Include controls for protein synthesis inhibition (cyclohexamide) and appropriate cell identification methods

How do G2E3 expression patterns correlate with cancer cell sensitivity to DNA-damaging agents?

Understanding the relationship between G2E3 expression and cancer cell sensitivity to DNA-damaging agents is critical for potential therapeutic applications:

• Baseline Expression Correlation:

  • Research indicates that G2E3 depletion sensitizes cancer cells to DNA damage, suggesting that higher G2E3 expression may contribute to chemoresistance

  • Methodologically, researchers should establish baseline G2E3 expression across cancer cell lines via quantitative RT-PCR and protein analysis before testing drug sensitivity

• Dynamic Regulation During Treatment:

  • Studies show that G2E3 mRNA and protein levels decrease following treatment with various DNA-damaging agents (cisplatin, gemcitabine, neocarzinostatin)

  • This downregulation appears to be part of the cellular response to DNA damage

  • Researchers should monitor the kinetics of G2E3 downregulation in relation to cell death markers to establish temporal relationships

• Mechanism of Sensitization:

  • G2E3 depletion accelerates apoptosis upon cisplatin treatment, as evidenced by increased levels of cleaved caspase 3 and cleaved PARP-1

  • When studying gemcitabine treatment, G2E3 knockdown increases replicative stress as measured by single-stranded DNA accumulation

  • These mechanisms should be evaluated using appropriate markers in combination with G2E3 antibodies

• p53-Dependent and Independent Effects:

  • Interestingly, while G2E3 regulates p53 levels, its depletion sensitizes both p53-positive and p53-negative cells to DNA damage

  • This suggests multiple mechanisms of action that researchers should investigate using isogenic cell lines differing only in p53 status

• Correlation with Clinical Outcomes:

  • Future research should investigate whether G2E3 expression levels in patient samples correlate with response to DNA-damaging chemotherapeutics

  • Researchers could employ tissue microarrays and immunohistochemistry with validated G2E3 antibodies for such studies

What methodological approaches can distinguish between G2E3's roles in ubiquitination versus other cellular functions?

Distinguishing between G2E3's ubiquitination activity and its other potential functions requires specialized methodological approaches:

• Domain-Specific Mutant Analysis:

  • Create and express G2E3 mutants with inactivated HECT domain (the catalytic domain for ubiquitination)

  • Compare these with wild-type G2E3 and other domain mutants in functional assays

  • Use G2E3 antibodies to confirm expression levels and localization of mutant proteins

• Ubiquitination Assays:

  • Perform in vitro ubiquitination assays with purified components including recombinant G2E3

  • Conduct cellular ubiquitination assays using HA-tagged ubiquitin and G2E3 antibodies to immunoprecipitate potential substrates

  • Compare ubiquitination patterns in cells with normal versus depleted G2E3 levels

• Target Identification:

  • Combine G2E3 immunoprecipitation with mass spectrometry to identify interacting proteins

  • Validate potential substrates using targeted ubiquitination assays

  • Perform proteomic analysis of ubiquitinated proteins in control versus G2E3-depleted cells

• Functional Rescue Experiments:

  • Deplete endogenous G2E3 and express either wild-type or ubiquitination-deficient mutants

  • Determine which cellular phenotypes can be rescued by which constructs

  • This approach can separate ubiquitination-dependent functions from other potential roles

• Localization Studies:

  • G2E3 is a nucleo-cytoplasmic shuttling protein

  • Use compartment-restricted G2E3 mutants (e.g., the nuclear export deficient mutant GFP-G2E3(2-363))

  • Determine how restricted localization affects both ubiquitination activity and other cellular functions

How should researchers integrate G2E3 antibody data with genomic and transcriptomic approaches for comprehensive pathway analysis?

To achieve comprehensive pathway analysis, researchers should integrate G2E3 antibody-based data with genomic and transcriptomic approaches using the following methodological strategies:

• Multi-Omics Experimental Design:

  • Design experiments that simultaneously collect samples for protein analysis (using G2E3 antibodies), RNA sequencing, and potentially chromatin studies

  • Ensure consistent experimental conditions and time points across different analytical platforms

  • Include appropriate perturbations (G2E3 knockdown, DNA damage treatments) that reveal functional relationships

• Correlation Analysis Between Protein and mRNA Levels:

  • Compare G2E3 protein levels (detected with antibodies) with mRNA expression (measured by qRT-PCR or RNA-seq)

  • Research has shown that both G2E3 mRNA and protein decrease upon DNA damage, suggesting transcriptional regulation

  • Identify conditions where protein and mRNA levels diverge, potentially indicating post-transcriptional regulation

• Network Analysis Integration:

  • Combine protein interaction data (from co-immunoprecipitation with G2E3 antibodies) with transcriptional changes following G2E3 depletion

  • Use pathway enrichment analysis to identify biological processes affected by G2E3

  • Construct regulatory networks integrating G2E3 protein interactions and transcriptional effects

• Temporal Resolution Studies:

  • Perform time-course experiments tracking G2E3 protein levels, localization, and associated changes in the transcriptome

  • This approach can establish cause-effect relationships and regulatory hierarchies

  • Particular attention should be paid to the kinetics of G2E3 downregulation following DNA damage and subsequent cellular responses

• Functional Validation of Integrated Models:

  • Use targeted interventions (e.g., modulating predicted upstream regulators or downstream effectors of G2E3)

  • Confirm model predictions using G2E3 antibodies to monitor protein responses

  • Employ genetic approaches (CRISPR/Cas9 editing, siRNA) to validate key nodes in the integrated network