GINS2 Antibody

What is GINS2 and why is it important in cellular biology?

GINS2 (Go-Ichi-Ni-San 2), also known as PSF2 (Partner of Sld Five 2), is a critical component of the GINS complex that plays an essential role in DNA replication. In humans, GINS2 is a 185 amino acid protein with a molecular weight of approximately 21.4 kDa . It functions primarily in the nucleus and chromosomes as part of the CDC45-MCM-GINS (CMG) helicase complex, the molecular machine that unwinds template DNA during replication and around which the replisome is built .

The biological significance of GINS2 lies in its role in both the initiation of DNA replication and the progression of DNA replication forks . Without proper GINS2 function, cells cannot effectively replicate their DNA, which impacts cell cycle progression and genomic stability. The collaboration of GINS2 with other proteins in the replication complex ensures successful DNA unwinding and synthesis, demonstrating its critical role in cell proliferation and genomic stability .

What are the most validated applications for GINS2 antibodies in research?

GINS2 antibodies have been validated for multiple research applications, with Western Blot (WB) being the most widely used and extensively validated technique. According to published literature and product data, GINS2 antibodies have been successfully employed in:

• Western Blot (WB): Most commercial antibodies show strong validation for detecting GINS2 at approximately 25 kDa (observed molecular weight)

• Immunohistochemistry (IHC-P): Used for detection in paraffin-embedded tissue sections

• Immunofluorescence (IF/ICC): Validated for cellular localization studies

• Flow Cytometry (FC): Used for intracellular detection

• ELISA: Some antibodies are suitable for enzyme-linked immunosorbent assays

Research publications have predominantly cited Western Blot applications, with at least 11 publications specifically using this method for GINS2 detection .

What cell types and tissues show significant GINS2 expression?

GINS2 expression has been documented in multiple cell types and tissues, with notable patterns in both normal and pathological samples:

Cancer cell lines with confirmed GINS2 expression:

• SKOV-3 and OVCAR3 (ovarian cancer cell lines)

• Jurkat cells (T-cell leukemia)

• HeLa cells (cervical cancer)

• HepG2 cells (liver cancer)

Tissue expression patterns:

• High expression in epithelial ovarian cancer (EOC) tissues (58.33% of samples) compared to normal ovarian tissues (16.67%)

• Strong nuclear localization in carcinoma cells

• Weak expression in normal ovarian tissue, with some signals in blood vessels

• Altered expression in peripheral blood of patients with intervertebral disk degeneration

This expression profile makes GINS2 a valuable research target for understanding both normal cellular processes and pathological conditions, particularly in oncology research.

What are the recommended protocols for detecting GINS2 in different experimental applications?

Western Blot Protocol:

• Recommended dilution: 1:1000-1:4000

• Expected molecular weight: 21 kDa (calculated), typically observed at 25 kDa

• Sample preparation: Total protein extraction from cells/tissues with standard lysis buffer containing protease inhibitors

• Loading control: β-actin is commonly used as internal reference

• Detection method: ECL reagent (Thermo Scientific Pierce) has been successfully used

Immunohistochemistry Protocol:

• Recommended dilution: 1:50-1:500

• Antigen retrieval: TE buffer pH 9.0 (alternatively, citrate buffer pH 6.0)

• Positive control tissues: Pancreatic cancer tissue has shown reliable staining

• Visualization: DAB chromogen with hematoxylin counterstain

Immunofluorescence/ICC Protocol:

• Recommended dilution: 1:50-1:500

• Cell fixation: 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100

• Blocking: 5% BSA in PBS

• Nuclear counterstain: DAPI

Flow Cytometry Protocol:

• Recommended amount: 0.25 μg per 10^6 cells in 100 μl suspension

• Permeabilization required for intracellular staining

How can I validate the specificity of a GINS2 antibody for my particular application?

Validating antibody specificity is crucial for reliable results. For GINS2 antibodies, consider these validation approaches:

• Genetic validation:

  • Use GINS2 knockdown cells (shRNA approach has been documented in multiple studies)

  • Compare antibody signal between wild-type and GINS2-depleted samples

  • Expected result: >90% signal reduction in knockdown samples

• Expression validation:

  • Use cell lines with known GINS2 expression (Jurkat, HeLa, HepG2, or SKOV-3)

  • Expected result: Detection at approximately 25 kDa in Western blot

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide/protein

  • Expected result: Signal abolishment or significant reduction

• Multiple antibody validation:

  • Use antibodies targeting different epitopes of GINS2

  • Expected result: Similar detection pattern across antibodies

• Molecular weight verification:

  • Ensure detection at expected molecular weight (21-25 kDa)

  • Note any post-translational modifications that might affect migration

What are the critical factors affecting GINS2 antibody performance in Western blots?

Several factors can significantly impact GINS2 antibody performance in Western blot applications:

• Sample preparation:

  • Complete protein denaturation is essential

  • Use fresh samples when possible

  • Include protease inhibitors in lysis buffer to prevent degradation

• Antibody dilution optimization:

  • Titrate antibody concentration (recommended starting range: 1:1000-1:4000)

  • Different lots may require adjustment of dilution factors

• Blocking conditions:

  • Typically 5% non-fat milk or BSA in TBST

  • Some antibodies may perform better with specific blocking agents

• Incubation conditions:

  • Primary antibody: Overnight at 4°C yields best results

  • Secondary antibody: 1 hour at room temperature (typically 1:2000 dilution)

• Detection method sensitivity:

  • ECL reagents of appropriate sensitivity for the expected expression level

  • Exposure time optimization to avoid saturation

• Post-translational modifications:

  • GINS2 undergoes multiple modifications including phosphorylation (S104, T158, S179) and ubiquitination (K70, K73, K80, K109, K118)

  • These modifications may affect antibody binding or protein migration

How is GINS2 expression altered in cancer, and how can antibodies help investigate this?

GINS2 expression alterations have been documented in multiple cancer types, with antibody-based detection methods playing a crucial role in these discoveries:

Cancer types with altered GINS2 expression:

• Epithelial ovarian cancer (EOC): 58.33% of EOC tissues showed high GINS2 expression compared to 16.67% in normal ovarian tissues

• Breast cancer: GINS2 transcript highly expressed, with expression levels correlating with histological grade (17.2% in grade 1, 50% in grade 2, and 77.1% in grade 3)

• Lung adenocarcinoma: Identified as a differentially expressed gene (high expression) in stage II

• Colon cancer: Overexpressed compared to normal tissues

• Gastric adenocarcinoma, pancreatic cancer: Carcinogenic role reported

Antibody applications in cancer research:

• Tissue expression profiling: IHC with GINS2 antibodies can reveal expression patterns across different tumor grades and stages

• Prognostic marker evaluation: Correlating GINS2 expression with patient outcomes

• Mechanistic studies: When combined with functional assays following GINS2 knockdown or overexpression

• Therapeutic response monitoring: Evaluating GINS2 expression changes following treatment

Research has demonstrated that manipulating GINS2 expression levels (through knockdown or overexpression) affects cancer cell behavior, suggesting its potential as a therapeutic target .

What is the role of GINS2 in intervertebral disk degeneration, and how was this discovered?

An intriguing and unexpected role for GINS2 was discovered in intervertebral disk degeneration (IDD), where its expression pattern differs from that observed in cancer:

Key findings on GINS2 in IDD:

• GINS2 is significantly downregulated in the peripheral blood of patients with intervertebral disk degeneration

• This was initially discovered through bioinformatics analysis of the GEO database (GSE124272) and subsequently validated in clinical samples

• ROC curve analysis yielded a high AUC of 0.8261 (95% CI = 0.7051-0.9472), suggesting GINS2 could serve as a potential diagnostic biomarker for IDD

Functional significance in nucleus pulposus cells:

• Overexpression of GINS2 increased proliferation, migration, and invasion of nucleus pulposus (NP) cells

• GINS2 overexpression decreased apoptotic properties of NP cells

• These findings suggest GINS2 might be a potential therapeutic target for IDD

The discovery process involved:

• Initial bioinformatics screening of differentially expressed genes

• Validation in peripheral blood samples from 30 IDD patients and 30 healthy participants

• Functional studies in NP cells using GINS2 overexpression plasmids

• Evaluation of cellular behaviors using proliferation, migration, invasion, and apoptosis assays

What methodological approaches can determine if GINS2 expression correlates with clinical outcomes?

To investigate correlations between GINS2 expression and clinical outcomes, researchers should consider the following methodological approaches:

• Tissue microarray (TMA) analysis:

  • Use validated GINS2 antibodies (1:50-1:500 dilution for IHC)

  • Score staining intensity and percentage of positive cells

  • Large patient cohorts with complete clinical data are essential

  • Example scoring system: High expression defined as positive staining in >50% of cells with moderate-to-strong intensity

• Peripheral blood analysis:

  • RNA extraction followed by RT-qPCR for GINS2 mRNA quantification

  • Protein analysis using ELISA or Western blot with GINS2 antibodies

  • Standardized collection protocols to minimize pre-analytical variables

• Statistical analysis approaches:

  • Receiver operating characteristic (ROC) curves to assess diagnostic potential (as demonstrated for IDD with AUC = 0.8261)

  • Kaplan-Meier survival analysis stratified by GINS2 expression levels

  • Cox proportional hazards regression for multivariate analysis

  • Correlation analysis with established clinical parameters

• Longitudinal sample collection:

  • Baseline and follow-up samples to track expression changes over disease progression

  • Correlation with treatment response and disease recurrence

• Multi-omics integration:

  • Combine GINS2 protein expression data with genomic, transcriptomic, and clinical data

  • Pathway enrichment analysis (such as KEGG and GO databases) to understand biological context

How does post-translational modification of GINS2 affect antibody selection and experimental design?

GINS2 undergoes multiple post-translational modifications (PTMs) that can significantly impact antibody binding and experimental outcomes:

Documented PTMs of GINS2:

Implications for antibody selection:

• Epitope consideration: Select antibodies whose epitopes do not include or overlap with known PTM sites if detecting total GINS2 is the goal

• Modification-specific antibodies: For studying specific PTMs, choose antibodies that specifically recognize phosphorylated or ubiquitinated forms

• Multiple antibody approach: Use antibodies targeting different regions to ensure comprehensive detection

Experimental design considerations:

• Sample preparation:

  • Include phosphatase inhibitors when studying phosphorylated forms

  • Include deubiquitinating enzyme inhibitors when studying ubiquitinated forms

  • Consider lambda phosphatase treatment to remove phosphorylation if it interferes with detection

• Migration pattern awareness:

  • PTMs can alter the apparent molecular weight in SDS-PAGE

  • The calculated molecular weight is 21 kDa, but the observed weight is often 25 kDa

  • Multiple bands may represent different modification states

• Cell cycle considerations:

  • GINS2 phosphorylation may vary throughout the cell cycle

  • Cell synchronization might be necessary for studying specific modifications

What are the key differences between polyclonal and monoclonal GINS2 antibodies, and when should each be used?

Both polyclonal and monoclonal GINS2 antibodies are available for research, each with distinct characteristics that make them suitable for different applications:

Polyclonal GINS2 antibodies:

• Generated in rabbits (most common)

• Target multiple epitopes across the GINS2 protein

• Examples: Proteintech 16247-1-AP, Abcam ab197123, Affinity Biosciences DF9451

• Applications: Validated for WB, IHC, IF/ICC

• Advantages:

  • Higher sensitivity due to recognition of multiple epitopes

  • More robust to minor protein denaturation or fixation effects

  • Better for detection of low abundance targets

• Best used for:

  • Initial protein characterization

  • Detection in various species (many have cross-reactivity)

  • Applications requiring high sensitivity

Monoclonal GINS2 antibodies:

• Generated in mice or rabbits

• Target a single epitope on GINS2

• Applications: Typically more specific for particular applications

• Advantages:

  • Consistent lot-to-lot reproducibility

  • Higher specificity for a single epitope

  • Lower background in some applications

• Best used for:

  • Studies requiring high reproducibility

  • Specific epitope targeting

  • Applications where background is problematic

Selection guide based on research needs:

• For novel research areas or initial characterization: Start with polyclonal antibodies for broader detection capabilities

• For reproducible, long-term studies: Consider monoclonal antibodies once the detection parameters are established

• For targeting specific forms/regions: Select antibodies with appropriate epitope specificity

• For cross-species studies: Check validated reactivity (human and mouse are most commonly validated)

How can researchers troubleshoot inconsistent results when using GINS2 antibodies in different experimental systems?

When encountering inconsistent results with GINS2 antibodies across different experimental systems, consider this systematic troubleshooting approach:

1. Antibody validation issues:

• Verify antibody specificity using positive and negative controls

• Consider using GINS2 knockdown or knockout systems as negative controls

• Test multiple antibodies targeting different epitopes

• Check lot-to-lot variation by requesting Certificate of Analysis

2. Sample preparation factors:

• Protein extraction method: Different buffers may affect epitope accessibility

• Fixation conditions (for IHC/IF): Overfixation can mask epitopes

• Protein denaturation (for WB): Ensure complete denaturation with heat and reducing agents

• Sample storage: Degradation due to improper storage or freeze-thaw cycles

3. Technical parameters to optimize:

• Antibody dilution: Titrate across a wider range than recommended

• Incubation conditions: Time and temperature adjustments

• Blocking reagents: Test alternative blocking solutions (BSA vs. milk)

• Detection systems: Try more sensitive detection methods

4. Biological variables to consider:

• Cell cycle stage: GINS2 expression and localization may vary across the cell cycle

• Cell confluence: Growth conditions can affect expression levels

• Post-translational modifications: Different conditions may alter modification patterns

• Species differences: Check if the antibody is validated for your species of interest

5. Systematic optimization approach:

• Change only one variable at a time

• Document all conditions thoroughly

• Include internal controls in each experiment

• Consider positive controls from published studies (e.g., Jurkat, HeLa, or SKOV-3 cells)

6. Application-specific troubleshooting:

• For WB: Try different membrane types, transfer conditions, or detection systems

• For IHC: Test multiple antigen retrieval methods

• For IF: Adjust permeabilization conditions

• For Flow cytometry: Optimize fixation and permeabilization protocols

How can GINS2 antibodies be used to investigate the protein's role in the DNA replication complex?

GINS2 antibodies can serve as powerful tools for dissecting the protein's function within the DNA replication complex through several advanced approaches:

Co-immunoprecipitation (Co-IP) studies:

• Use GINS2 antibodies to pull down the entire GINS complex and associated proteins

• Can identify interaction partners in the CDC45-MCM-GINS (CMG) helicase complex

• Western blot analysis of immunoprecipitates can confirm known interactions or identify novel binding partners

• Mass spectrometry analysis of immunoprecipitates can provide unbiased identification of the complete interactome

Chromatin immunoprecipitation (ChIP) assays:

• Use GINS2 antibodies to identify genomic regions where the GINS complex is bound

• Can map GINS2 localization at replication origins and forks

• ChIP-seq approaches provide genome-wide binding profiles

• Time-course experiments can track GINS2 movement during S phase

Proximity ligation assays (PLA):

• Visualize protein-protein interactions in situ

• Combine GINS2 antibodies with antibodies against other replication factors

• Can reveal spatial and temporal dynamics of interactions

Immunofluorescence co-localization:

• Use GINS2 antibodies alongside markers for replication forks (e.g., PCNA, RPA)

• Can track assembly and disassembly of replication complexes

• Live-cell imaging with fluorescently tagged antibody fragments can monitor dynamics

Cell cycle synchronization experiments:

• Track GINS2 expression, localization, and complex formation throughout the cell cycle

• Compare normal cells with cancer cells where replication may be dysregulated

• Combine with inhibitors of cell cycle checkpoints to dissect regulatory mechanisms

What are the latest findings regarding GINS2's potential as a therapeutic target, and how can antibodies contribute to this research?

Emerging research suggests GINS2 may have significant potential as a therapeutic target in various diseases, with antibodies playing a crucial role in advancing this field:

Current therapeutic potential findings:

• In cancer therapy:

  • GINS2 knockdown inhibits proliferation and induces apoptosis in multiple cancer types

  • High expression correlates with cancer progression in breast cancer, lung adenocarcinoma, and EOC

  • Involvement in cancer cell migration and invasion suggests potential for targeting metastasis

• In intervertebral disk degeneration (IDD):

  • Downregulated in IDD patient blood samples

  • Overexpression promotes nucleus pulposus cell proliferation and migration

  • Potential therapeutic approach may involve restoring GINS2 levels in IDD

Antibody contributions to therapeutic development:

• Target validation:

  • Use of GINS2 antibodies in tissue microarrays to confirm expression in patient samples

  • IHC analysis to correlate expression with disease progression and patient outcomes

  • Western blot and qPCR validation of knockdown efficiency in functional studies

• Mechanism elucidation:

  • Pathway analysis using phospho-specific antibodies to determine downstream effectors

  • Co-IP studies to identify protein-protein interactions that could be therapeutically targeted

  • Subcellular localization studies to understand compartment-specific functions

• Therapeutic antibody development:

  • While GINS2's nuclear localization makes it challenging for direct antibody targeting, antibodies can guide development of other therapeutic modalities

  • Antibody-based screening assays for small molecule inhibitors

  • Validation of target engagement in drug development

• Biomarker applications:

  • Diagnostic potential demonstrated by ROC analysis (AUC = 0.8261 for IDD)

  • Patient stratification for clinical trials

  • Monitoring treatment response

Future research directions:

• Development of cell-penetrating antibodies or antibody fragments targeting GINS2

• Combining GINS2 targeting with standard therapies

• Exploration of synthetic lethality approaches in cancers with high GINS2 expression

How do GINS2 expression patterns differ between normal development, tissue regeneration, and pathological states?

Understanding the differential expression of GINS2 across various physiological and pathological contexts can provide valuable insights into its biology and potential as a therapeutic target:

GINS2 in normal development and homeostasis:

• Expression in actively proliferating tissues due to its role in DNA replication

• Present in embryonic tissues with high replicative potential

• Low or undetectable levels in most adult differentiated tissues

• Detectable in adult tissues with high cellular turnover (bone marrow, intestinal epithelium)

• Conserved across species, with orthologs reported in mouse, rat, bovine, frog, zebrafish, chimpanzee, and chicken

GINS2 in tissue regeneration and repair:

• Potential upregulation during wound healing and tissue regeneration

• In nucleus pulposus cells, GINS2 overexpression promotes proliferation, suggesting a role in tissue repair mechanisms

• Likely activated during liver regeneration due to high replicative demands

GINS2 in pathological states:

• Cancer:

  • Significantly upregulated in multiple cancer types

  • Expression correlates with histological grade in breast cancer (17.2% in grade 1, 50% in grade 2, and 77.1% in grade 3)

  • Nuclear localization in cancer cells

  • Functional studies indicate it promotes cancer cell proliferation and inhibits apoptosis

• Intervertebral disk degeneration:

  • Downregulated in peripheral blood of IDD patients

  • Experimental overexpression increases proliferation of nucleus pulposus cells

  • May represent a compensatory mechanism or contribute to pathology

• Other degenerative conditions:

  • Potential dysregulation in conditions with aberrant cellular proliferation

  • May be involved in fibrotic disorders characterized by excessive cell proliferation

Experimental approaches to study differential expression:

• Comparative IHC studies:

  • Use GINS2 antibodies on tissue microarrays containing normal, regenerating, and pathological samples

  • Quantitative scoring of expression levels and subcellular localization

• Single-cell analysis:

  • Combine GINS2 antibodies with cell-type-specific markers

  • Flow cytometry or mass cytometry (CyTOF) for quantitative analysis

  • Single-cell RNA-seq with protein validation using antibodies

• Developmental time-course studies:

  • Track GINS2 expression during embryonic development and postnatal growth

  • Compare with expression during tissue regeneration models

• 3D organoid cultures:

  • Study GINS2 expression and function in developing organoids

  • Compare normal versus disease-mimicking conditions

This comprehensive understanding of context-dependent GINS2 expression can guide more precise therapeutic targeting and biomarker development strategies.