Biological Background of CENPI

Centromere Protein I (CENPI) is a core component of the Constitutive Centromere-Associated Network (CCAN), a multi-subunit protein complex that resides permanently at the centromere throughout the cell cycle. CENPI functions as part of the CENP-H/I/K sub-complex within the CCAN, playing a critical role in establishing and maintaining a functional kinetochore — the macromolecular structure that mediates attachment of chromosomes to spindle microtubules during mitosis and meiosis.

CENPI is essential for accurate chromosome segregation. Disruption of CENPI expression leads to misalignment of chromosomes at the metaphase plate, increased rates of lagging chromosomes during anaphase, and ultimately chromosomal instability (CIN). Because CIN is a hallmark of many human cancers, CENPI has attracted significant interest as a potential biomarker and research target in oncology. Recombinant Human CENPI Protein enables researchers to perform mechanistic in vitro studies that would be difficult or impossible using endogenous protein from cell lysates alone.

Handling and Reconstitution Protocol

Proper handling of Recombinant Human CENPI Protein is critical to preserving its structural integrity and functional activity. Follow the steps below upon receipt and before each use:

• Upon receipt: Inspect the vial for integrity. Briefly centrifuge the vial (1,000–2,000 × g, 10–15 s) to collect any residual material from the cap and walls before opening.
• Reconstitution: If supplied in lyophilized form, reconstitute by adding the volume of sterile, deionized water (or the buffer specified on the Certificate of Analysis) indicated on the datasheet. Allow the vial to stand at room temperature for 5–10 minutes without vortexing; gently swirl to dissolve completely. Avoid vigorous agitation, which can cause protein aggregation or denaturation.
• Working concentration: Prepare working aliquots at concentrations suitable for your assay. As a general guideline, dilute in a carrier protein-containing buffer (e.g., PBS + 0.1% BSA) to reduce non-specific adsorption to tube surfaces.
• Aliquoting: Prepare single-use aliquots immediately after reconstitution to minimize repeated freeze-thaw cycles, which can significantly reduce protein activity.

Note: Specific reconstitution volumes, buffer compositions, and recommended working concentrations should be verified against the product-specific Certificate of Analysis (CoA) provided with each lot.

Storage Conditions and Stability

Correct storage of Recombinant Human CENPI Protein is essential for maintaining its biological activity over time.

• Long-term storage: Store lyophilized protein at -20°C to -80°C. Once reconstituted, aliquots should be stored at -80°C.
• Short-term storage: Reconstituted protein that will be used within 1–2 weeks may be stored at 4°C; however, addition of a carrier protein (0.1% BSA) and a protease inhibitor cocktail is strongly recommended to reduce degradation.
• Freeze-thaw cycles: Limit freeze-thaw cycles to an absolute minimum — ideally no more than three — as each cycle can lead to protein aggregation, loss of tertiary structure, and reduced functional activity.
• Stability: Under recommended storage conditions, lyophilized protein is generally stable for 12 months from the date of manufacture. Reconstituted protein stability is shorter; refer to the CoA for lot-specific expiry information.

Always refer to the product datasheet and CoA for lot-specific storage recommendations, as formulation may vary between production lots.

Research Applications

Recombinant Human CENPI Protein is suited to a range of in vitro and cell-based research applications, including:

• Protein–protein interaction studies: CENPI interacts with multiple CCAN members (e.g., CENP-H, CENP-K, CENP-L, CENP-M, CENP-N). Recombinant CENPI can be used in co-immunoprecipitation (co-IP), pull-down assays, and surface plasmon resonance (SPR) to map binding interfaces and measure interaction affinities.
• Western blot (WB): Recombinant CENPI serves as a positive control antigen for validating anti-CENPI antibodies and optimizing Western blot protocols.
• ELISA and immunoassay development: Use as a coating antigen or capture standard to develop and calibrate sandwich ELISA kits for CENPI quantification.
• Structural biology: Suitable as a component for crystallography or cryo-electron microscopy studies of CCAN sub-complexes, subject to purity and formulation requirements.
• Chromosome segregation and mitosis research: Can be used in cell-free systems or microinjection experiments to probe the functional consequences of altered CENPI levels on mitotic fidelity.
• Drug discovery / target validation: Useful for screening small-molecule or peptide inhibitors targeting CENPI–CENP-H/K interactions relevant to oncology research.

Quality Control and Purity Considerations

When working with any recombinant protein, verifying quality before use is best practice. For Recombinant Human CENPI Protein, researchers should confirm the following from the Certificate of Analysis (CoA):

• Purity: Recombinant proteins intended for biochemical assays should typically be ≥85% pure as assessed by SDS-PAGE and/or HPLC. Confirm the purity specification for your specific lot from the CoA.
• Endotoxin level: For cell-based assays, endotoxin contamination must be below acceptable thresholds (typically <1.0 EU/μg by LAL assay). Verify the endotoxin specification on the CoA.
• Identity confirmation: Identity should be confirmed by mass spectrometry or N-terminal sequencing as stated on the CoA.
• Activity: Where a functional activity assay is available (e.g., binding to CENP-H/K partners), confirm the reported specific activity meets the requirements of your downstream assay format.
• Lot-to-lot variability: Record the lot number used in all experiments and publications to enable reproducibility. Contact Aladdin Scientific technical support if inter-lot variability is observed.