CINP Antibody

What is CINP and why are antibodies against it important in research?

CINP (cyclin dependent kinase 2 interacting protein) is a human protein with a molecular mass of approximately 24.3 kilodaltons that plays significant roles in genome maintenance and DNA damage response pathways. Antibodies against CINP are critical research tools for studying cell cycle regulation, DNA damage responses, and genome stability mechanisms. These antibodies enable detection, quantification, and localization of CINP in various experimental systems, providing insights into its function in normal and pathological conditions. Understanding CINP's interactions, particularly with the ATR-ATRIP complex, has significant implications for research on genomic integrity and cellular responses to replication stress .

What types of CINP antibodies are available for research applications?

Research-grade CINP antibodies are available in multiple formats including monoclonal and polyclonal variants with various species reactivities. The most commonly available are unconjugated antibodies that recognize human CINP, though mouse and rat cross-reactive antibodies are also available. Additionally, specialized conjugated variants (HRP-conjugated, biotin-conjugated) exist for specific applications. The antibodies vary in their validated applications, with most being optimized for Western blot (WB), immunohistochemistry (IHC), immunofluorescence (IF), and immunoprecipitation (IP) techniques . When selecting an antibody, researchers should consider the specific epitope recognized, as some antibodies target the N-terminal region of CINP while others recognize other domains of the protein .

How should CINP antibodies be stored and handled to maintain optimal activity?

For optimal performance, CINP antibodies should be stored according to manufacturer recommendations, typically at -20°C for long-term storage with aliquoting to prevent freeze-thaw cycles. Working dilutions can generally be kept at 4°C for 1-2 weeks. Prior to use in experiments, antibodies should be centrifuged briefly to collect liquid at the bottom of the tube. When preparing dilutions, use high-quality, filtered buffers appropriate for the application (e.g., TBST for Western blotting, PBS with carrier protein for immunofluorescence). Since CINP antibodies are used to detect relatively low-abundance proteins involved in genome maintenance pathways, proper handling is critical to maintain sensitivity. Always include appropriate positive controls (e.g., cell lines known to express CINP) and negative controls (e.g., CINP-depleted samples) in experimental designs to validate antibody performance .

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

CINP antibodies serve as powerful tools for investigating DNA damage response (DDR) pathways due to CINP's role as an ATR-ATRIP interacting protein. For comprehensive analysis of DDR pathways, researchers can employ co-immunoprecipitation assays using anti-CINP antibodies to isolate protein complexes and identify interacting partners through immunoblotting with additional antibodies, including those against ATRIP, ATR, and other DDR proteins. This approach has confirmed that Flag-CINP binds endogenous ATRIP and that CINP interacts with the ATR-ATRIP complex, although this interaction appears to be transient or low-affinity .

To investigate CINP's role in replication stress response, researchers can combine CINP antibody staining with treatment protocols using hydroxyurea (HU) or other replication stress inducers. Unlike some DDR proteins, CINP shows pan-nuclear staining without concentration into ATRIP-containing foci following DNA damage, suggesting it may function through transient interactions rather than as part of stable damage-associated complexes. For optimal results in these experiments, use cell fixation methods that preserve nuclear architecture and protein-protein interactions, such as paraformaldehyde fixation for immunofluorescence studies .

What methodological approaches can be used to study CINP-ATRIP interactions using antibodies?

Several complementary approaches can be employed to study CINP-ATRIP interactions:

• Co-immunoprecipitation (Co-IP): Use anti-CINP antibodies to pull down protein complexes from cell lysates, followed by Western blotting with anti-ATRIP antibodies. The reciprocal experiment using anti-ATRIP antibodies for IP and anti-CINP for detection can also provide valuable insights. Evidence shows that Flag-CINP binds endogenous ATRIP, confirming this interaction .

• Proximity Ligation Assay (PLA): This technique can detect protein-protein interactions in situ by generating fluorescent signals when two antibodies (anti-CINP and anti-ATRIP) bind targets in close proximity (<40 nm).

• Domain Mapping: Based on yeast two-hybrid studies, CINP interacts with the N-terminal half of the ATRIP coiled-coil domain (specifically amino acids 118-156). Researchers can design experiments using deletion mutants of ATRIP (e.g., ΔATRIP coiled-coil domain) to confirm that removing this region eliminates CINP interaction .

• Functional Studies: Since the ATRIP coiled-coil domain is required for ATRIP dimerization, stable ATR binding, ATRIP accumulation at DNA lesions, and ATR-dependent checkpoint signaling, researchers can assess how CINP affects these processes using specific antibodies against phosphorylated checkpoint proteins .

How can CINP antibodies be utilized in genome maintenance screening assays?

CINP antibodies can be incorporated into high-throughput screening assays for genome maintenance genes and pathways using the following methodological approaches:

• Immunofluorescence-based Screens: Using automated microscopy platforms, researchers can conduct large-scale screens where cells treated with different genetic perturbations (e.g., siRNA libraries) are stained with CINP antibodies alongside markers of DNA damage (γH2AX) or replication stress (pRPA).

• Protein Complex Analysis: CINP antibodies can be used to isolate protein complexes via immunoprecipitation, with subsequent mass spectrometry analysis to identify novel interaction partners. This approach complements yeast two-hybrid screens, which have already identified ATRIP as a CINP-interacting protein .

• ChIP-seq Applications: For investigating CINP's potential association with chromatin, CINP antibodies can be employed in chromatin immunoprecipitation assays coupled with next-generation sequencing to map genomic binding sites.

• Functional Genomic Validation: Following initial screens that identify CINP as a genome maintenance gene, antibodies against CINP provide a means to validate knockdown efficiency in follow-up experiments using immunoblotting techniques. This is particularly important given CINP's role in DNA damage response and hydroxyurea sensitivity phenotypes .

What are the optimal conditions for using CINP antibodies in Western blot applications?

For optimal Western blot results with CINP antibodies, follow these methodological guidelines:

• Sample Preparation: Extract proteins using RIPA or NP-40 buffer supplemented with protease and phosphatase inhibitors. Since CINP is a nuclear protein involved in DNA damage responses, include benzonase or other nucleases in your lysis buffer to ensure efficient extraction from chromatin-associated fractions.

• Gel Electrophoresis: Use 10-12% polyacrylamide gels for optimal resolution of CINP (24.3 kDa). Load appropriate positive controls, such as HeLa cell lysates which express detectable levels of endogenous CINP.

• Transfer Conditions: For efficient transfer of CINP, use PVDF membranes with 0.2 μm pore size and transfer at 100V for 1 hour or 30V overnight.

• Blocking and Antibody Incubation:

  • Block membranes with 5% non-fat milk or BSA in TBST for 1 hour at room temperature

  • Dilute primary anti-CINP antibodies according to manufacturer recommendations (typically 1:1000 to 1:2000)

  • Incubate with primary antibody overnight at 4°C with gentle agitation

  • Wash extensively with TBST (4 x 5 minutes)

  • Incubate with appropriate HRP-conjugated secondary antibody (1:5000-1:10000) for 1 hour at room temperature

  • Develop using enhanced chemiluminescence detection

• Expected Results: CINP should appear as a band at approximately 24.3 kDa. Validate antibody specificity using CINP-depleted cell lysates (siRNA or CRISPR) as negative controls.

What considerations are important when using CINP antibodies for immunoprecipitation studies?

When designing immunoprecipitation experiments with CINP antibodies, consider these methodological aspects:

How should researchers optimize CINP antibody dilutions for immunofluorescence microscopy?

For successful immunofluorescence detection of CINP, follow these optimization guidelines:

• Fixation Protocol: Test both paraformaldehyde (4%, 10-15 minutes) and methanol (-20°C, 10 minutes) fixation, as the optimal method may depend on epitope accessibility. Based on previous studies showing pan-nuclear staining of CINP, paraformaldehyde fixation followed by Triton X-100 permeabilization often provides good results .

• Antibody Dilution Series: Perform a titration experiment starting with the manufacturer's recommended dilution and testing 2-fold dilutions above and below this concentration (typically starting around 1:100-1:500).

• Blocking Optimization: Test different blocking solutions (3-5% BSA, normal serum, or commercial blocking reagents) to minimize background staining.

• Signal Enhancement: For low-abundance proteins like CINP, consider tyramide signal amplification or similar methods to enhance detection sensitivity.

• Controls: Include appropriate controls:

  • Positive control: Cell lines known to express CINP

  • Negative control: CINP-depleted samples (siRNA or CRISPR knockout)

  • Secondary antibody-only control: To assess background fluorescence

• Expected Staining Pattern: CINP typically shows pan-nuclear staining without concentration into damage-induced foci. Unlike some DNA damage response proteins, CINP does not form discrete foci in response to DNA damage or replication stress, which is consistent with its proposed role in transient interactions with the ATR-ATRIP complex .

How can researchers validate the specificity of CINP antibody staining patterns?

Validating CINP antibody specificity requires a multi-faceted approach:

• Genetic Depletion Controls: The gold standard for antibody validation is comparing staining between wild-type samples and those where the target protein has been depleted. For CINP:

  • Use siRNA or shRNA to knockdown CINP expression

  • Create CRISPR/Cas9 knockout cell lines

  • Compare staining patterns or band intensities between control and depleted samples

• Multiple Antibody Validation: Use at least two different CINP antibodies that recognize distinct epitopes. Consistent staining patterns or Western blot bands across different antibodies strongly support specificity.

• Recombinant Protein Controls: For Western blot applications, include lanes with purified recombinant CINP protein as positive controls.

• Peptide Competition Assays: Pre-incubate the CINP antibody with excess immunizing peptide before staining. Specific staining should be significantly reduced or eliminated.

• Correlation with Expression Data: Staining intensity should correlate with known expression levels across different cell types or experimental conditions.

• Expected Patterns: Based on published data, specific CINP staining should show predominantly nuclear localization without concentration in discrete foci after DNA damage, unlike some other DNA damage response proteins .

What are common issues when using CINP antibodies for co-immunoprecipitation of ATRIP complexes?

Researchers may encounter several challenges when attempting to co-immunoprecipitate CINP-ATRIP complexes:

• Low Co-IP Efficiency: The interaction between CINP and ATR-ATRIP is likely transient or low affinity, as studies have shown that only a small percentage of these proteins co-immunoprecipitate. To improve detection:

  • Use chemical crosslinking prior to lysis

  • Optimize lysis conditions to preserve weak interactions

  • Increase antibody concentration and incubation time

  • Consider using epitope-tagged constructs (Flag-CINP has been successfully used)

• Non-specific Binding: To reduce background:

  • Pre-clear lysates with protein A/G beads

  • Use more stringent washing buffers

  • Include appropriate blocking proteins (BSA or non-immune serum)

  • Use proper controls (isotype-matched non-specific antibodies)

• Detection Sensitivity: Since endogenous interactions may be difficult to detect:

  • Use sensitive detection methods (ECL Prime or Femto)

  • Consider concentrating eluted proteins

  • Optimize exposure times for Western blots

• Buffer Composition Effects: The CINP-ATRIP interaction is not stimulated by DNA damage or replication stress, so experimental conditions should be optimized accordingly. Avoid harsh detergents that might disrupt protein-protein interactions .

How should researchers interpret discrepancies in CINP detection between different antibody-based techniques?

When facing discrepancies in CINP detection across different techniques, consider these methodological explanations and solutions:

• Epitope Accessibility Differences:

  • Western Blot: Denatured proteins expose all epitopes

  • Immunofluorescence: Fixed, native conformation may mask some epitopes

  • Immunoprecipitation: Native, folded state may hide linear epitopes

  Solution: Use antibodies against different regions of CINP or try different fixation/preparation methods.

• Protein Complex Masking:

  • CINP's interaction with ATRIP via its coiled-coil domain may mask antibody binding sites in native conditions

  • In some applications, CINP may be part of protein complexes that obscure epitopes

  Solution: Use epitope mapping data to select antibodies targeting regions not involved in protein interactions.

• Post-translational Modifications:

  • Some antibodies may have reduced affinity for phosphorylated or otherwise modified CINP forms

  Solution: Characterize antibody recognition of modified CINP forms or use modification-insensitive antibodies.

• Detection Sensitivity Thresholds:

  • Western blotting may detect lower protein amounts than immunofluorescence

  Solution: Apply signal amplification methods for less sensitive techniques.

• Sample Preparation Variables:

  • Nuclear proteins like CINP require efficient extraction methods

  Solution: Optimize extraction protocols with nucleases, higher salt concentrations, or specialized nuclear extraction buffers .

How does CINP function in the ATR-ATRIP pathway based on antibody-mediated studies?

Based on antibody-mediated studies, CINP functions in the ATR-ATRIP pathway through the following mechanisms:

• Direct ATRIP Interaction: Co-immunoprecipitation experiments using anti-Flag antibodies have demonstrated that Flag-CINP binds endogenous ATRIP. The reciprocal immunoprecipitation confirms this interaction. Additionally, an interaction between endogenous ATR-ATRIP and CINP has been observed, although the interaction appears to be transient or low affinity .

• ATRIP Binding Domain Specificity: Through a combination of yeast two-hybrid screening and immunoprecipitation studies using antibodies against various ATRIP constructs, researchers have mapped the CINP binding region to the N-terminal half of the predicted ATRIP coiled-coil domain (amino acids 118-156). This domain is critical for ATRIP function, as it mediates dimerization, stable ATR binding, accumulation at DNA lesions, and checkpoint signaling .

• Regulation Independent of DNA Damage: Antibody-based experiments have shown that the CINP-ATR-ATRIP interaction is not stimulated by DNA damage or replication stress. Furthermore, subcellular localization studies using CINP antibodies revealed pan-nuclear staining without concentration into ATRIP-containing foci in response to DNA damage. These findings suggest CINP may regulate ATR-ATRIP via transient interactions and is unlikely to be part of the active signaling complex at DNA damage sites .

• Functional Role: Functional genomic screens identified CINP as a genome maintenance gene, with cells showing sensitivity to hydroxyurea and DNA damage when CINP is depleted. The interaction with the ATR-ATRIP complex suggests CINP may function in the replication stress response pathway, potentially by modulating ATR-ATRIP complex formation or activity .

What experimental approaches using CINP antibodies can help elucidate its role in replication stress response?

To investigate CINP's role in replication stress response, researchers can employ these antibody-based experimental approaches:

• Chromatin Fractionation with Immunoblotting:

  • Separate cellular fractions (cytoplasmic, nucleoplasmic, chromatin-bound)

  • Immunoblot with CINP antibodies to determine subcellular localization after replication stress

  • Compare CINP distribution to known replication stress response proteins (RPA, ATR, ATRIP)

• Checkpoint Activation Analysis:

  • Treat cells with hydroxyurea or other replication stress inducers

  • Immunoblot for phosphorylated ATR substrates (Chk1-P, RPA-P) in CINP-depleted versus control cells

  • Use CINP antibodies to confirm knockdown efficiency

• Proximity Ligation Assays (PLA):

  • Perform PLA using antibodies against CINP and various replication fork proteins

  • Compare PLA signals before and after replication stress induction

  • Quantify interaction frequencies in different cell cycle phases

• Chromatin Immunoprecipitation (ChIP):

  • Use CINP antibodies for ChIP to identify genomic loci where CINP binds

  • Perform after replication stress to determine if CINP associates with stalled forks

  • Combine with nascent DNA labeling techniques to focus on replication-associated regions

• iPOND (isolation of Proteins On Nascent DNA) with CINP Detection:

  • Use this technique to purify proteins associated with replication forks

  • Immunoblot with CINP antibodies to determine if CINP associates with active or stalled forks

  • Compare to ATRIP association patterns

How can researchers design experiments to identify novel CINP-interacting proteins beyond ATRIP?

To discover novel CINP interaction partners beyond ATRIP, researchers can implement these methodological approaches:

• Co-Immunoprecipitation coupled with Mass Spectrometry:

  • Use validated anti-CINP antibodies to immunoprecipitate endogenous CINP

  • Process samples for mass spectrometry analysis

  • Include appropriate controls (IgG IP, CINP-depleted samples)

  • Perform under various conditions (normal, replication stress, DNA damage)

  • Use quantitative proteomics (SILAC, TMT) to identify condition-specific interactions

• BioID or TurboID Proximity Labeling:

  • Generate CINP fusion with biotin ligase (BioID2 or TurboID)

  • Express in cells and add biotin to label proximal proteins

  • Purify biotinylated proteins and identify by mass spectrometry

  • Validate hits with reciprocal co-immunoprecipitation using antibodies against candidate interactors

• Yeast Two-Hybrid Screening:

  • Use full-length CINP or specific domains as bait

  • Screen against human cDNA libraries

  • Validate hits in mammalian cells using co-immunoprecipitation with specific antibodies

  • Previous Y2H screens using CINP identified ATRIP as an interactor, confirming the validity of this approach

• Protein Complex Immunoprecipitation:

  • Use antibodies against known CINP-interacting proteins (e.g., ATRIP)

  • Perform sequential immunoprecipitation (first with anti-ATRIP, then with anti-CINP)

  • Identify proteins present in both single and sequential IPs

  • Compare protein complexes under different cellular conditions