RO-3306, IC-01248880
Product Introduction
RO-3306 is a potent, selective, ATP-competitive, and reversible small-molecule inhibitor of cyclin-dependent kinase 1 (CDK1) with an IC₅₀ of 20 nM and a Ki of 35 nM. It exhibits greater than 10-fold selectivity for CDK1 over a panel of related cyclin-dependent kinases (CDK2, CDK4, CDK5, CDK7, CDK9), making it a highly specific pharmacological tool for dissecting CDK1-dependent cell cycle regulation. RO-3306 reversibly arrests proliferating cells at the G2/M boundary by preventing CDK1/cyclin B1 complex activation, thereby blocking mitotic entry without inducing DNA damage. Upon compound washout, cells synchronously re-enter the cell cycle and progress through mitosis, enabling reversible synchronization of cell populations in G2 phase for coordinated biochemical and cell biological analyses. RO-3306 has been extensively utilized in cell cycle research, cancer biology, and high-content screening applications to investigate mitotic entry mechanisms, checkpoint regulation, and CDK1-dependent phosphorylation networks.
Product Features
1. Potent and Selective CDK1 Inhibitor: Inhibits CDK1 with an IC₅₀ of 20 nM and displays greater than 10-fold selectivity over other CDKs, minimizing off-target kinase effects.
2. Reversible G2/M Arrest: Reversibly blocks cells at the G2/M boundary without inducing DNA damage; upon inhibitor washout, cells synchronously progress through mitosis.
3. ATP-Competitive and Cell-Permeable: Readily crosses cell membranes for effective CDK1 inhibition in live-cell assays.
4. DNA Damage-Free Synchronization: Enables reversible synchronization of cell populations in G2 phase without genotoxic stress associated with traditional synchronization methods.
5. High Purity: Supplied at 98% purity for reproducible pharmacological studies.
Specifications
Size: Available in 1 mg, 5 mg, 1 mL (10 mM in DMSO), 10 mg, 50 mg, 100 mg, and 1 g
CAS Number: 872573-93-8
Purity: 98%
Molecular Weight: 301.73
Molecular Formula: C18H15ClN2O
Solubility: DMSO: ¡Ý15 mg/mL; limited solubility in aqueous buffers
Storage and Stability
Storage Conditions: Store at 2-8¡ãC, sealed in dry conditions, and keep in a dark place. Protect from light and moisture. For the 10 mM DMSO solution, store at -20¡ãC protected from light.
Shelf Life: The product is stable for 24 months from the date of manufacture when stored as directed. For reconstituted solutions in DMSO, aliquot into single-use volumes and store at -20¡ãC or -80¡ãC. Avoid repeated freeze-thaw cycles.
Protocol (For Reference Only)
Important: RO-3306 is light-sensitive and hygroscopic. Protect from light and moisture during handling and storage. Prepare stock solutions in anhydrous DMSO.
1. Stock Solution Preparation (Solid): Dissolve RO-3306 in anhydrous DMSO to prepare a concentrated stock solution (e.g., 10-50 mM). Aliquot into single-use vials and store at -20¡ãC protected from light. The 10 mM ready-to-use DMSO solution may be used directly for dilution into culture medium.
2. In Vitro Working Solution Preparation: Dilute DMSO stock solution into pre-warmed complete culture medium to achieve a final concentration of 9 µM for G2/M arrest. Limit final DMSO concentration in culture medium to ¡Ü0.1% to avoid solvent-related cytotoxicity.
3. G2/M Synchronization: Add RO-3306 to proliferating cells at a final concentration of 9 µM and incubate for 18-20 hours to achieve complete G2/M arrest. Monitor cell cycle distribution by propidium iodide staining with flow cytometry or by assessing mitotic markers (phospho-histone H3) by immunofluorescence or Western blot.
4. Reversal and Mitotic Entry: Following G2/M arrest, wash cells twice with pre-warmed PBS to remove the inhibitor, add fresh complete medium without RO-3306, and monitor synchronized mitotic entry by time-lapse microscopy or by collecting samples at defined time points for cell cycle and biochemical analyses.
5. CDK1 Inhibition Validation: Assess target engagement by measuring CDK1 kinase activity in immunoprecipitates from treated cells, or evaluate CDK1 substrate phosphorylation (e.g., lamin A/C, histone H1) by Western blot or phospho-specific antibodies.
Precautions
1. RO-3306 is light-sensitive and hygroscopic; always store sealed in dry conditions at 2-8¡ãC and protect from light.
2. The optimal concentration for G2/M arrest is 9 µM for most cell lines; however, titration between 6-12 µM may be necessary for specific cell types. Prolonged exposure (>24 hours) or concentrations above 10 µM may induce apoptosis in certain cell lines.
3. Upon washout, cells rapidly enter mitosis; prepare all materials in advance for time-sensitive analyses of mitotic events.
4. RO-3306-mediated G2/M arrest is reversible and does not induce DNA damage, making it superior to traditional synchronization methods using nocodazole or DNA-damaging agents; however, extended arrest may cause some cells to escape and re-enter the cycle.
5. For research use only. Not for use in diagnostic or therapeutic procedures.
FAQ (Simplified)
Q1: What is the recommended concentration for G2/M cell cycle synchronization?
A1: RO-3306 at 9 µM for 18-20 hours effectively arrests most cell lines at the G2/M boundary. Titration between 6-12 µM may be required for specific cell types. Verify arrest by flow cytometry or phospho-histone H3 staining.
Q2: How does RO-3306 compare to other synchronization methods?
A2: Unlike nocodazole (microtubule disruption) or DNA-damaging agents, RO-3306 induces reversible G2/M arrest without causing DNA damage or microtubule perturbation. This makes it the preferred method for studying unperturbed mitotic entry and CDK1-dependent processes.
Q3: How quickly do cells enter mitosis after RO-3306 washout?
A3: Cells typically begin entering mitosis within 15-30 minutes after washout, with peak mitotic index occurring at 45-90 minutes depending on cell type. Prepare time-lapse or time-course sampling protocols accordingly.
Q4: Does RO-3306 inhibit other kinases?
A4: RO-3306 displays greater than 10-fold selectivity for CDK1 over CDK2, CDK4, CDK5, CDK7, and CDK9. It also shows minimal activity against a broad panel of non-CDK kinases. However, at concentrations above 20 µM, off-target effects may be observed.
Disclaimer
1. For Research Use Only. Not for use in diagnostic or therapeutic procedures.
2. Due to the variable nature of biological research, optimization of conditions using a small-scale pilot experiment is strongly recommended before processing valuable samples.
3. This warranty is limited to the replacement of the product. The manufacturer assumes no liability for incidental or consequential damages, including loss of samples or data.
4. Wear appropriate protective clothing and gloves when handling this product.
Ordering Information
Catalog Number: IC-01248880
Product Name: RO-3306
Size: 1 mg / 5 mg / 1 mL (10 mM in DMSO) / 10 mg / 50 mg / 100 mg / 1 g
Price:
1 mg: CNY ¥500.00 / USD $50.00 / EUR €60.00 / JPY ¥9000.00
5 mg: CNY ¥1000.00 / USD $100.00 / EUR €120.00 / JPY ¥18000.00
1 mL (10 mM in DMSO): CNY ¥1100.00 / USD $110.00 / EUR €132.00 / JPY ¥19800.00
10 mg: CNY ¥1390.00 / USD $139.00 / EUR €166.80 / JPY ¥25020.00
50 mg: CNY ¥3780.00 / USD $378.00 / EUR €453.60 / JPY ¥68040.00
100 mg: CNY ¥6050.00 / USD $605.00 / EUR €726.00 / JPY ¥108900.00
1 g: CNY ¥32800.00 / USD $3280.00 / EUR €3936.00 / JPY ¥590400.00
2023 Version
