CORI3 Antibody

What is the CORI3 antibody and what cellular processes does it target?

CORI3 antibody targets the I3 protein, which functions as a single-stranded DNA binding protein (SSB) essential in various cellular processes. The I3 protein demonstrates striking affinity for single-stranded DNA while showing minimal binding to double-stranded DNA . This protein plays a critical role in DNA replication and repair mechanisms, with evidence suggesting it serves as an important SSB involved in these processes . Structurally, I3 contains conserved patterns of aromatic and charged amino acids commonly found in replicative SSBs, allowing it to interact with DNA in a salt-resistant manner with approximately 10 nucleotides per binding site .

What are the recommended purification methods for CORI3 antibody?

Based on established antibody purification protocols, CORI3 antibody can be effectively purified using HiTrap protein A column chromatography from cell supernatants. For experimental applications requiring Fab fragments:

• Begin with purified IgG at 10 mg/ml concentration

• Perform proteolytic digestion using immobilized papain

• Remove Fc fragments using protein A column chromatography

• Further purify by gel filtration chromatography on a Superdex 200 16/60 column

• Collect and concentrate the elution peak corresponding to approximately 50 kDa molecular mass

This multi-step purification process ensures high purity antibody preparations suitable for crystallization trials and other sensitive applications requiring minimal contaminants.

How should CORI3 antibody be stored to maintain activity?

While specific storage conditions for CORI3 antibody aren't directly addressed in the search results, established protocols for maintaining antibody activity include:

• Store purified antibody at -20°C to -80°C for long-term storage

• For working solutions, maintain at 4°C with sodium azide (0.02%) as a preservative

• Avoid repeated freeze-thaw cycles which can diminish binding capacity

• If using for crystallization studies, maintain freshly purified preparations at 4°C without freezing

How can CORI3 antibody be used to investigate protein-DNA interactions?

CORI3 antibody can be used to study I3 protein interactions with DNA through several advanced techniques:

• Electrophoretic Mobility Shift Assays (EMSA): The I3 protein shows distinctive binding characteristics with single-stranded DNA that can be measured through EMSA. Unlike cooperative binding seen with some SSBs, I3 protein exhibits low cooperativity and can bind numerous molecules to a template, creating a stoichiometric interaction pattern .

• Fluorescence Resonance Energy Transfer (FRET) analysis: As demonstrated with other antibodies, FRET-based competition assays can be developed using donor-labeled and acceptor-labeled Fab fragments to precisely assess epitope binding and competition in solution. This approach eliminates avidity effects or steric hindrance outside the antigen-antibody binding interface .

• Salt Resistance Testing: Since I3 protein demonstrates salt-resistant DNA binding, experiments can be designed to compare binding under varying salt concentrations to distinguish specific from non-specific interactions.

What approaches can be used to assess CORI3 antibody epitope specificity?

Epitope characterization for CORI3 antibody can be performed using several complementary approaches:

• X-ray Crystallography: Crystal structures of antibody-antigen complexes provide high-resolution mapping of epitope binding sites. For example, the structures of N60-i3 Fab-gp120 complexes were resolved at 3.2Å resolution using molecular replacement techniques, allowing precise epitope mapping .

• Competition Binding Assays: Using FRET-FCS (Fluorescence Resonance Energy Transfer-Fluorescence Correlation Spectroscopy) competition assays to determine if CORI3 antibody competes with known antibodies for binding sites. This method helps identify overlapping epitopes and binding characteristics .

• Mutagenesis Studies: Systematic mutation of potential binding sites on the target protein can identify critical residues involved in antibody recognition. This approach has been used successfully to map epitopes for other antibodies like C11 .

• Epitope Binning: Group antibodies based on their competition patterns to identify those recognizing similar or distinct epitopes on the target protein.

How can CORI3 antibody be used to investigate protein phosphorylation states?

The I3 protein is phosphorylated on serine residues post-infection , making CORI3 antibody potentially valuable for studying phosphorylation states. Researchers can:

• Develop phospho-specific variants of CORI3 antibody that selectively recognize phosphorylated vs. non-phosphorylated forms of I3

• Use CORI3 antibody in combination with phosphatase treatments to distinguish contribution of phosphorylation to protein function

• Apply techniques similar to those used for studying Aurora B phosphorylation states:

  • Immunofluorescence with phospho-specific antibodies to monitor spatial distribution of phosphorylated protein

  • Western blotting with phospho-specific antibodies to quantify total cellular levels of phosphorylated vs. unphosphorylated protein

What controls should be included when using CORI3 antibody in immunofluorescence studies?

When designing immunofluorescence experiments with CORI3 antibody, include the following controls:

• Primary Antibody Specificity Control: Include samples where primary antibody is omitted or replaced with isotype-matched control antibody

• siRNA Validation: As demonstrated with I3 protein studies, siRNA-mediated depletion can validate antibody specificity. Create an siRNA-resistant variant of the target protein tagged with GFP to confirm specificity through rescue experiments

• Peptide Competition: Pre-incubation of antibody with excess purified peptide should abolish specific staining

• Correlation with Live-Cell Markers: When possible, correlate fixed-cell immunofluorescence results with live-cell data using fluorescent protein-tagged constructs

How can CORI3 antibody be optimized for immunoprecipitation experiments?

For optimal immunoprecipitation results:

• Cross-linking Optimization: Determine optimal antibody cross-linking conditions to protein A/G beads to minimize antibody leaching and contamination in IP samples

• Buffer Compatibility Testing: Test multiple lysis buffer compositions to identify conditions that preserve antibody-antigen interaction while effectively solubilizing the target protein

• Validation Strategy:

  • Prepare control and I3-depleted cell extracts

  • Perform parallel IPs from both samples

  • Compare recovery of target protein and interacting partners

  • Confirm specific interactions through reciprocal IPs with antibodies against suspected interacting proteins

What approaches can resolve contradictory data when using CORI3 antibody?

When facing contradictory results:

• Complementary Detection Methods: If contradictory data appears between different experimental approaches (e.g., between localization studies and biochemical assays), implement multiple detection methods:

  • Combine immunofluorescence with biochemical fractionation

  • Use proximity ligation assays to verify protein-protein interactions in situ

  • Apply FRET-based approaches to validate interactions in living cells

• Re-evaluation of Prior Studies: As demonstrated in the research on PP1γ localization, where contradictory findings to previous reports were observed , carefully re-examine experimental conditions that might explain discrepancies:

  Study	PP1γ Localization Finding	Experimental Conditions
  Posch et al., 2010	Loss of GFP-PP1γ from kinetochores	SDS22 or I3 depletion
  Current study	No loss of GFP-PP1γ from kinetochores	SDS22 or I3 depletion compared to control or NIPP1 depletion

• Antibody Validation: Evaluate if contradictions stem from antibody cross-reactivity or epitope masking by:

  • Testing multiple antibody clones targeting different epitopes

  • Comparing antibody binding under native vs. denaturing conditions

  • Validating with genetic approaches (knockout/knockdown)

How can CORI3 antibody be used to study protein-protein interactions?

CORI3 antibody can be employed in several advanced approaches to study protein-protein interactions:

• Co-immunoprecipitation coupled with Mass Spectrometry: Use CORI3 antibody to pull down I3 protein complexes and identify interacting partners through mass spectrometry analysis

• Proximity-dependent Labeling: Combine with BioID or APEX techniques by creating fusion proteins to identify proteins in close proximity to I3 in living cells

• Sequential Immunoprecipitation: For analyzing multi-protein complexes, perform sequential IPs as demonstrated in studies of the PP1-SDS22-I3 complex:

  Based on research findings, I3 appears to form a complex with PP1 and SDS22, where I3 specifically sequesters the SDS22-PP1 complex and prevents its association with kinetochores . This regulatory mechanism suggests a model where:

  • SDS22 binds PP1 in solution

  • I3 regulates this interaction

  • This regulation affects PP1 recruitment to specific cellular locations

What methodologies can integrate CORI3 antibody with high-throughput screening approaches?

CORI3 antibody can be integrated into high-throughput screening through:

• Yeast Surface Display Systems: As mentioned in antibody engineering studies , yeast surface display combined with fluorescence-activated cell screening can be used for antibody optimization or epitope mapping in high-throughput format

• Automated Microscopy Platforms: Implement CORI3 antibody in high-content screening to analyze subcellular localization changes under various treatment conditions:

  • Design 96/384-well format immunofluorescence protocols

  • Combine with automated image analysis for quantification

  • Correlate localization patterns with functional outcomes

• Phage Display Libraries: Generate and screen phage display libraries with variants of the target epitope to identify critical binding determinants for CORI3 antibody

How can researchers quantitatively assess CORI3 antibody binding characteristics?

For quantitative assessment of binding characteristics:

• Surface Plasmon Resonance (SPR): Determine binding kinetics (kon, koff) and equilibrium constants (KD) using purified recombinant proteins

• Isothermal Titration Calorimetry (ITC): Measure thermodynamic parameters of binding (ΔH, ΔS, ΔG)

• FRET-FCS Analysis: As demonstrated for other antibodies, this technique provides precise assessment of binding competition in solution :

  Parameter	Methodology	Key Advantages
  Binding Competition	FRET-FCS between donor-labeled and acceptor-labeled Fabs	Eliminates avidity effects and steric clashes outside binding interface
  Binding Site Size	Electrophoretic mobility shift assays	Determines number of nucleotides covered by protein binding
  Cooperativity	Quantitative binding isotherms	Assesses whether binding of one molecule affects binding of subsequent molecules

How can non-specific binding be reduced when using CORI3 antibody in immunofluorescence?

To minimize non-specific binding:

• Optimized Blocking Protocol:

  • Test various blocking agents (BSA, normal serum, commercial blockers)

  • Determine optimal blocking time and temperature

  • Consider pre-adsorption of antibody with cell/tissue extracts from knockout/knockdown samples

• Antibody Dilution Series:

  • Perform systematic dilution series to identify concentration with optimal signal-to-noise ratio

  • Compare staining pattern across multiple cell types and fixation methods

• Signal Validation:

  • Confirm specificity through siRNA depletion rescue experiments as demonstrated for I3 protein

  • Use multiple antibodies targeting different epitopes of the same protein

What strategies can resolve inconsistencies in CORI3 antibody performance between applications?

When antibody performance varies between applications:

• Buffer Optimization:

  • Different applications may require different buffer conditions

  • Systematically test buffer components (salt concentration, detergents, pH)

  • Consider effects of fixation on epitope accessibility

• Epitope Accessibility Analysis:

  • Native vs. denatured applications may yield different results

  • Test epitope accessibility under various conditions using peptide arrays

  • Consider native immunoprecipitation followed by western blotting to validate antibody recognition across methods

• Batch Validation:

  • Validate each antibody lot in all intended applications

  • Maintain reference samples for comparison across experiments

  • Consider monoclonal alternatives if polyclonal variability is an issue

How can CORI3 antibody be validated for knockout or knockdown studies?

For rigorous validation in genetic depletion studies:

• siRNA Rescue Experiments: As demonstrated for I3 protein , generate siRNA-resistant variants of the target protein. This approach allows:

  • Confirmation that observed phenotypes are due to specific protein depletion

  • Validation of antibody specificity through detection of the rescue construct

  • Structure-function studies through rescue with mutant variants

• Titration Analysis:

  • Perform partial knockdowns to establish dose-dependent relationships

  • Correlate protein levels (by western blot) with observed phenotypes

  • Compare antibody detection with mRNA levels by qRT-PCR

• Cross-validation with Genome Editing:

  • Compare siRNA/shRNA results with CRISPR/Cas9 knockout models

  • Analyze multiple independent clones or knockdown constructs

  • Include appropriate controls for off-target effects

How can CORI3 antibody studies be integrated with structural biology approaches?

Integration with structural biology can be achieved through:

• X-ray Crystallography: Similar to the approach used for N60-i3 Fab complex , CORI3 antibody Fab fragments can be co-crystallized with purified target protein to determine:

  • Precise epitope binding sites

  • Structural changes induced by antibody binding

  • Mechanistic insights into protein function

• Cryo-electron Microscopy: For larger complexes or membrane-associated targets, cryo-EM can provide structural information at near-atomic resolution

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): This approach can map antibody binding sites through differential solvent accessibility in the presence/absence of antibody

What considerations are important when designing CORI3 antibody-based functional assays?

When designing functional assays:

• Temporal Resolution: Consider the dynamics of the biological process being studied:

  • For cell cycle studies like those examining I3 function in mitosis , synchronize cells and sample at appropriate intervals

  • Use live-cell imaging with fluorescently tagged markers to correlate with fixed-cell antibody staining

• Spatial Resolution: For proteins with discrete subcellular localization:

  • Combine with subcellular fractionation to correlate biochemical and imaging data

  • Consider super-resolution microscopy techniques for detailed localization studies

  • Use proximity ligation assays to verify specific interactions in situ

• Functional Readouts: Design assays with clear functional endpoints:

  • For DNA binding proteins like I3 , measure DNA replication efficiency or repair capacity

  • For mitotic regulators, assess chromosome alignment and mitotic timing

How can researchers adapt CORI3 antibody for use in advanced immunological techniques?

CORI3 antibody can be adapted for specialized immunological applications:

• Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assays: Similar to those described for N60-i3 and JR4 antibodies , CORI3 antibody can be tested for ADCC activity using:

  • Target cells sensitized with appropriate antigens

  • Peripheral blood mononuclear effector cells

  • Flow cytometry-based cytolysis detection methods

• Single-Cell Analysis Platforms: Integrate with:

  • Mass cytometry (CyTOF) for multiplexed protein detection

  • Single-cell RNA-seq to correlate protein levels with transcriptional states

  • High-throughput single-cell next-generation sequencing as mentioned in antibody discovery approaches

• Translational Modification Detection: Develop assays to detect specific post-translational modifications, such as the serine phosphorylation observed in I3 protein , using phospho-specific antibody variants.