CRIP2 Antibody

What is CRIP2 and what are its key structural features?

CRIP2, also known as CRP2, ESP1, or cardiac LIM protein, is a 208-amino acid protein with a predicted molecular weight of 23 kDa. Its gene is mapped to human chromosome 14q32.3. CRIP2 contains two LIM domains that form a paired zinc finger structure separated by two amino acid linkers with the consensus sequences (CXXCX17HXXC) and (CXXCX17CHXXC) . These LIM domains are crucial for mediating protein-protein or protein-DNA interactions, which are essential for the biological functions of numerous LIM proteins . The protein shares 93% amino acid sequence identity with its rat homolog (Esp1 or Crp2) .

What is the tissue distribution pattern of CRIP2?

Northern blot analysis has revealed widespread tissue expression of the 1.3-kb CRIP2 mRNA, with the highest expression levels found in the heart. Moderate expression occurs in the lung, brain, placenta, spleen, and kidney, while other tissues and organs show relatively low expression . Interestingly, in testis, a second 1.7-kb mRNA has also been detected . Within the vascular system, CRIP2 is expressed predominantly in the posterior cardinal vein and caudal vein plexus intersegmental vein .

What are the primary biological functions of CRIP2?

CRIP2 serves multiple biological functions:

• Vascular development: CRIP2 controls migration, adhesion, and proliferation of endothelial cells by interacting with cytoskeleton proteins KRT8 and VIM, modulating the VEGFA/CDC42 signaling pathway, and affecting interaction with SRF through PDE10A/cAMP and PDGF/JAK/STAT/SRF signaling .

• Tumor suppression: CRIP2 acts as a transcription repressor of NF-κB–mediated proangiogenic cytokine expression, functionally inhibiting tumor formation and angiogenesis . It interacts with NF-κB/p65 to inhibit its DNA-binding ability to the promoter regions of major proangiogenesis cytokines (IL6, IL8, and VEGF) .

• Cardiovascular regulation: CRIP2 plays a crucial role in cardiac development and is involved in the pathological changes of cardiovascular diseases .

What criteria should be considered when selecting a CRIP2 antibody for research?

When selecting a CRIP2 antibody, researchers should evaluate:

• Antibody type: Polyclonal antibodies (like PA5-30997 and the rabbit polyclonal antibody) offer broad epitope recognition, while monoclonals provide greater specificity .

• Species reactivity: Confirm cross-reactivity with your experimental model. For example, some CRIP2 antibodies show predicted reactivity with mouse (92%), rat (92%), and bovine (93%) in addition to human .

• Application validation: Verify the antibody has been validated for your specific application (Western blot, ELISA, immunohistochemistry, etc.) .

• Positive controls: Use recommended positive controls for validation. A549 and HeLa cells are recommended positive controls for some CRIP2 antibodies .

• Recognition domain: Consider which domain of CRIP2 the antibody recognizes, particularly if studying domain-specific functions or interactions.

How should CRIP2 antibodies be validated before experimental use?

A rigorous validation protocol for CRIP2 antibodies should include:

• Western blot analysis: Confirm specificity by detecting a band at the expected molecular weight (~23 kDa). Use cell lines with known CRIP2 expression (A549, HeLa) as positive controls .

• Knockout/knockdown controls: Validate antibody specificity using CRIP2 knockout or knockdown models. CRISPR/Cas9 systems targeting intron/exon junctions 1, 2, and 6 of the Crip2 gene have been successfully employed to generate CRIP2 knockout models .

• Recombinant protein testing: If available, test the antibody against purified recombinant CRIP2 protein.

• Cross-reactivity assessment: Test for cross-reactivity with other CRIP family members (CRIP1, CRIP3) to ensure specificity, especially given their structural similarities .

• Immunoprecipitation validation: If using the antibody for IP experiments, verify its ability to immunoprecipitate CRIP2 using overexpression systems like CRIP2-Flag fusion proteins .

What are the optimal conditions for Western blot detection of CRIP2?

For optimal Western blot detection of CRIP2:

• Antibody concentration: Use anti-CRIP2 antibody at approximately 1.0 μg/mL concentration .

• Secondary antibody dilution: HRP-conjugated secondary antibody should be diluted 1:50,000-1:100,000 .

• Protein extraction: Use cold cell lysis buffer containing protease and phosphatase inhibitors to preserve protein integrity .

• Loading controls: Vinculin has been successfully used as a loading control in CRIP2 Western blots .

• Gel percentage: Use 10-12% polyacrylamide gels for optimal resolution of the 23 kDa CRIP2 protein.

• Membrane blocking: 5% non-fat milk or BSA in TBST is typically effective for blocking non-specific binding.

• Sample preparation: Centrifuge the antibody solution briefly prior to opening the vial, and store as a concentrated solution .

How can protein-protein interactions of CRIP2 be effectively studied?

To investigate CRIP2 protein-protein interactions:

• Coimmunoprecipitation (Co-IP):

  • Express CRIP2-Flag fusion protein in target cells (e.g., HUVECs)

  • Lyse cells in cold buffer containing protease and phosphatase inhibitors

  • Incubate with anti-Flag or control isotype IgG antibodies

  • Precipitate using protein A/G magnetic beads

  • Analyze via Western blot or mass spectrometry

• Recombinant protein Co-IP:

  • Clone human CRIP2 and potential interacting proteins (like KRT8, SRF, VIM) into vectors with different tags (Flag, HA)

  • Co-transfect into HEK293T cells

  • Perform Co-IP and analyze by Western blotting

• Proximity ligation assay: This method can provide in situ detection of protein-protein interactions with high specificity.

• Yeast two-hybrid screening: Useful for unbiased identification of novel CRIP2 interacting partners.

• Domain mapping: Generate constructs expressing specific CRIP2 domains to identify interaction interfaces.

What methodologies are recommended for studying CRIP2's role in vascular development?

To investigate CRIP2's role in vascular development:

• Zebrafish model: Zebrafish provide an excellent model for studying vascular development, allowing visualization of the posterior cardinal vein plexus and caudal vein plexus where CRIP2 is predominantly expressed .

• HUVEC functional assays:

  • Cell migration assays to evaluate VEGFA/CDC42 signaling effects

  • Adhesion assays to assess cytoskeleton formation and hyperadhesive phenotypes

  • Proliferation assays to measure effects on cell growth

• Signaling pathway analysis:

  • Examine the impact of CRIP2 on VEGFA/CDC42, PDE10A/cAMP, and PDGF/JAK/STAT/SRF signaling pathways

  • Use Western blotting, qPCR, and pathway inhibitors to dissect mechanisms

• Cytoskeleton visualization:

  • Immunofluorescence staining of KRT8 and VIM to assess CRIP2's effect on cytoskeleton formation

  • Quantitative analysis of cell morphology and cytoskeletal organization

• CRIP2 knockout/knockdown models:

  • CRISPR/Cas9 system targeting CRIP2 intron/exon junctions

  • Compare phenotypes in vascular structures between wild-type and CRIP2-deficient models

What are common challenges when working with CRIP2 antibodies and how can they be addressed?

Common challenges and solutions include:

• High background signal:

  • Increase blocking time or concentration (5-10% blocking agent)

  • Optimize antibody dilutions (start with 1:1000 for primary and adjust)

  • Include additional washing steps with higher stringency

  • Consider using different blocking agents (milk vs. BSA)

• Weak or no signal:

  • Verify CRIP2 expression in your sample; use positive controls (A549, HeLa)

  • Increase antibody concentration or incubation time

  • Use enhanced chemiluminescence detection systems

  • Consider concentrating proteins if expression is low

• Multiple bands/non-specific binding:

  • Increase antibody specificity by using monoclonal over polyclonal

  • Optimize washing conditions

  • Confirm specificity with knockout controls

  • Perform peptide competition assays

• Protein degradation:

  • Use fresh samples and keep on ice

  • Include appropriate protease inhibitors in lysis buffer

  • Store antibody as concentrated solution at -20°C or below, avoiding multiple freeze-thaw cycles

How can researchers optimize ELISA protocols for CRIP2 detection?

For ELISA optimization:

• Antibody dilution: Start with recommended dilution (1:62500 for some CRIP2 antibodies)

• Sample preparation: Standardize protein extraction procedures across experiments

• Blocking optimization: Test different blocking agents (BSA, milk, commercial blockers)

• Standard curve: Generate a standard curve using recombinant CRIP2 protein at known concentrations

• Incubation conditions: Optimize temperature and duration for each step

• Detection system: Select appropriate enzyme/substrate combinations for desired sensitivity

• Validation: Include positive and negative controls in each plate

• Cross-reactivity testing: Ensure specificity by testing against related proteins (CRIP1, CRIP3)

How can CRIP2 be targeted in cancer research, particularly regarding its tumor suppressor function?

Based on CRIP2's tumor suppressor functions:

• Functional complementation studies: Use microcell-mediated chromosome transfer (MMCT) approaches to introduce CRIP2 into cancer cell lines and assess tumor suppression effects .

• NF-κB pathway analysis: Investigate CRIP2's interaction with NF-κB/p65 and its inhibitory effect on DNA-binding ability to promoter regions of proangiogenesis cytokines (IL6, IL8, and VEGF) .

• Gene reexpression strategies: Develop methods to reexpress CRIP2 in tumor cells where it is downregulated, particularly in nasopharyngeal carcinoma and esophageal carcinoma cell lines .

• Mechanistic studies of tumor angiogenesis inhibition: Examine how CRIP2 functions as a transcription repressor of NF-κB–mediated proangiogenic cytokine expression .

• Biomarker development: Assess CRIP2 expression levels in tumor samples as a potential prognostic biomarker.

• Therapeutic targeting: Explore CRIP2-based therapeutic approaches for cancers where it is downregulated.

What are the latest methodological approaches to study CRIP2's role in cardiovascular pathologies?

Advanced methods for investigating CRIP2 in cardiovascular context:

• Single-cell RNA sequencing: Profile CRIP2 expression at the single-cell level in heart tissues to identify cell-specific functions.

• Conditional knockout models: Generate tissue-specific CRIP2 knockout mice to study its role in different cardiac cell types.

• Patient-derived organoids: Develop cardiac organoids from patient cells to study CRIP2 function in human disease contexts.

• CRISPR/Cas9 genome editing: Create specific mutations in CRIP2 LIM domains to investigate structure-function relationships .

• Proteomics approaches: Apply mass spectrometry-based proteomics to identify the complete CRIP2 interactome in cardiac cells.

• In vivo imaging: Use advanced imaging techniques to visualize CRIP2-dependent vascular development in real-time.

• ChIP-seq analysis: Identify genomic targets of CRIP2 to better understand its transcriptional regulatory functions.

How can researchers investigate the relationship between CRIP2 and other CRIP family members in experimental settings?

To study CRIP family member relationships:

• Comparative expression analysis: Quantify expression of CRIP1, CRIP2, and CRIP3 across tissues and under different conditions.

• Functional redundancy testing: Use knockout/knockdown of individual and multiple CRIP family members to assess compensatory mechanisms.

• Domain swapping experiments: Create chimeric proteins exchanging domains between CRIP family members to identify functional regions.

• Co-immunoprecipitation studies: Investigate potential heterodimerization or interaction between different CRIP family proteins.

• Transcriptional regulation analysis: Examine whether CRIP family members regulate each other's expression.

• Evolutionary conservation analysis: Compare CRIP family proteins across species to identify conserved and divergent functions.

• Systems biology approaches: Integrate data on all CRIP family members to model their functional interrelationships.