Background: CENPB Biology and Research Significance

Centromere Protein B (CENPB) is a highly conserved, sequence-specific DNA-binding protein that plays a fundamental role in centromere identity and kinetochore assembly. It recognizes and binds a 17-bp consensus sequence known as the CENP-B box, found within alpha-satellite repetitive DNA at human centromeres. CENPB is the only centromere protein known to bind centromeric DNA in a sequence-specific manner, making it a critical molecular anchor for the kinetochore complex.

Research involving CENPB is relevant to chromosome biology, cell division fidelity, and the study of centromere-associated diseases. Aberrant CENPB expression or function has been linked to chromosomal instability and has been investigated in the context of autoimmune conditions, where anti-CENP-B autoantibodies are a diagnostic marker for systemic sclerosis (scleroderma). Recombinant Human CENPB Protein enables researchers to conduct in vitro binding assays, generate and validate antibodies, and study protein-protein or protein-DNA interactions in a controlled biochemical environment.

Materials Required

• Recombinant Human CENPB Protein (SKU: rp365906, Aladdin Scientific)
• Sterile deionized water or manufacturer-recommended reconstitution buffer
• Microcentrifuge tubes (low-protein-binding, 1.5 mL)
• Micropipettes and calibrated tips
• Benchtop microcentrifuge
• Ice bucket or cold block
• Appropriate assay buffer (e.g., PBS, pH 7.4, with carrier protein if required)
• SDS-PAGE and Western blot apparatus (for quality verification)
• Protein quantification reagents (e.g., BCA or Bradford assay kit)
• -80°C freezer and -20°C freezer for storage

Note: Always consult the product datasheet supplied with SKU rp365906 for the exact reconstitution buffer, recommended concentration, and formulation details before beginning.

Step-by-Step Protocol: Reconstitution, Storage, and Quality Check

Step 1: Preparation

Remove the vial from -80°C storage and allow it to equilibrate briefly at room temperature (no more than 5 minutes) before opening. Keep the vial on ice throughout handling to minimize degradation.

Step 2: Reconstitution

Centrifuge the vial briefly (1,000 × g, 30 seconds) to collect lyophilized material at the bottom. Add the reconstitution buffer specified in the product datasheet slowly and directly onto the lyophilized pellet. Gently swirl or pipette to dissolve — do NOT vortex, as this may denature the protein. Allow the solution to stand on ice for 5–10 minutes until fully dissolved.

Step 3: Protein Quantification

Determine the protein concentration using a BCA or Bradford protein assay, referencing a BSA standard curve. Compare results with the expected concentration stated on the Certificate of Analysis (CoA). Acceptable recovery is typically ≥90% of the stated amount; flag significant deviations for review.

Step 4: Quality Verification by SDS-PAGE

Run an aliquot (typically 1–2 µg) under reducing SDS-PAGE conditions. Stain with Coomassie Blue or silver stain and verify that the band appears at the expected molecular weight (CENPB full-length human protein is approximately 80 kDa; confirm exact predicted MW from the datasheet for this recombinant construct). Purity should meet the specification stated on the CoA.

Step 5: Aliquoting and Storage

Divide the reconstituted protein into single-use aliquots in low-protein-binding tubes to avoid repeated freeze-thaw cycles. Snap-freeze aliquots in liquid nitrogen or dry ice/ethanol bath, then store at -80°C. For short-term use within 1–2 weeks, aliquots may be kept at -20°C. Adding a carrier protein (e.g., 0.1% BSA) to the storage buffer is recommended if diluting to very low concentrations to prevent adsorption to tube surfaces.

Application Guidelines

Recombinant Human CENPB Protein is suitable for a range of biochemical and cell biology applications. Recommended applications and general starting-point conditions are listed below; researchers should optimize conditions empirically for their specific experimental system.

• ELISA: Use as a coating antigen or standard for autoantibody detection assays, particularly for anti-CENP-B antibodies relevant to autoimmune disease research. Typical coating concentration range: 0.5–2 µg/mL (optimize per assay).
• Western Blot: Use as a positive control or molecular weight standard for CENPB detection. Load 50–200 ng per lane; confirm band at expected MW.
• Protein-DNA Interaction Studies (EMSA/gel shift): Use to study binding of CENPB to CENP-B box-containing oligonucleotides. Titrate protein concentration against labeled DNA substrate.
• Antibody Production and Validation: Use as immunogen or validation standard for raising and characterizing anti-CENPB antibodies.
• Kinetochore Assembly Assays: Use in reconstitution experiments to study CENPB interactions with other centromere/kinetochore components.

Important: Always include appropriate positive and negative controls in each experiment. Refer to the product datasheet for any application-specific notes.

Troubleshooting