5-Hydroxy-CTP |
Catalog No.GB20155
One-Click Copy Product Info
|
5-Hydroxy-CTP is a cytidine triphosphate containing a 5-hydroxy modification. 5-Hydroxy-CTP can serve as a monomeric substrate for in vitro mRNA transcription synthesis and can be incorporated into RNA chains to study epigenetic modifications.
Products are for research use only. Not for human use. We do not sell to patients.
Sample solution is provided at 25 µL, 10mM.
_1-3.$$$39978408@51.jpg');)
_1-9.$$$38538795@65.jpg');)
_1-9.$$$39112701@51.jpg');)
_1-7.$$$37316672@70.jpg');)
_366-372.$$$36198801@70.jpg');)
.$$$38565142@65.jpg');)
_1867-1883.$$$33248023@67.jpg');)
_eads6055.$$$39977546@45.jpg');)
_eadm9805.$$$40080571@45.jpg');)
_eadj3020.$$$39666821@45.jpg');)
_1-20.$$$39757301@28.jpg');)
_1-24.$$$38898113@28.jpg');)
_273-287.$$$36806353@46.jpg');)
_1-16.$$$37156877@44.jpg');)
_1-19.$$$37460805@44.jpg');)
_1291-1307.$$$34518654@46.jpg');)
5-Hydroxy-CTP is a cytidine triphosphate containing a 5-hydroxy modification. 5-Hydroxy-CTP can serve as a monomeric substrate for in vitro mRNA transcription synthesis and can be incorporated into RNA chains to study epigenetic modifications. 5-Hydroxy-CTP can be used in research related to gene expression regulation and RNA drug development[1-4].
References:
[1] Kawasaki K, Kanaba T, Yoneyama M, et al. Insights into substrate recognition by the Escherichia coli Orf135 protein through its solution structure. Biochem Biophys Res Commun. 2012 Apr 6;420(2):263-8.
[2] Kawasaki K, Yoneyama M, Murata-Kamiya N, et al. ¹H, ¹³C and ¹⁵N NMR assignments of the Escherichia coli Orf135 protein. Biomol NMR Assign. 2012 Apr;6(1):1-4.
[3] Malvezzi S, Sturla SJ, Tanasova M. Data in support of quantification of pyrophosphate as a universal approach to determine polymerase activity and assay polymerase inhibitors. Data Brief. 2015 Apr 22;4:14-8.
[4] Fujikawa K, Kasai H. The oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP, is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein. DNA Repair (Amst). 2002 Jul 17;1(7):571-6.
5-Hydroxy-CTP for Orf135 Protein Hydrolysis Activity Assay[1]
This protocol is adapted from research data and provided for reference only. Adjustments may be necessary based on specific experimental requirements.
1. Reagent Preparation: Dissolve 5-Hydroxy-CTP in water to prepare a stock solution of appropriate concentration (e.g., 5μM working solution); Prepare 20mM Tris-HCl buffer (pH 8.5); Prepare 10mM MgCl₂ solution; Prepare 80μg/ml bovine serum albumin (BSA) solution; Prepare 8mM dithiothreitol (DTT) solution; Prepare 10% glycerol solution; Prepare an anion-exchange HPLC column (TSK-GEL DEAE-2SW, 4.6mm × 250mm) and the corresponding mobile phase (50mM phosphate buffer, pH=7.0, containing 20% acetonitrile and 1mM EDTA).
2. Orf135 Protein Preparation: Clone the E. coli orf135 gene into the pGEX-6P-1 glutathione S-transferase (GST) fusion protein expression vector, and transform it into E. coli BL21 strain for expression. Purify the GST-Orf135 fusion protein through ammonium sulfate precipitation, glutathione Sepharose 4B affinity column chromatography, and gel filtration column chromatography. Remove the GST tag using protease and eliminate GST via a glutathione Sepharose 4B column to obtain recombinant Orf135 protein.
3. Pyrophosphatase Activity Assay: In a standard reaction mixture (100μl), add 20mM Tris-HCl (pH=8.5), 10mM MgCl₂, 80μg/ml BSA, 8mM DTT, 10% glycerol, and 5μM 5-Hydroxy-CTP substrate. Pre-incubate the mixture at 30°C for 2 minutes, add an appropriate amount of Orf135 protein (e.g., 0.2pmol for 5-Hydroxy-CTP assay), and incubate at 30°C for 5 minutes.
4. Reaction Termination and Product Analysis: Add 120μl of 10mM EDTA to terminate the reaction. Inject all samples into a anion-exchange column, and elute isocratically with 50mM phosphate buffer (pH=7.0), 20% acetonitrile, and 1mM EDTA at a flow rate of 1ml/min. Detect using an HPLC detection system at UV wavelength, and quantify nucleoside triphosphates and their hydrolyzed products (monophosphates) by measuring the UV absorbance peak area.
Precautions:
(1) The optimal pH for 5-Hydroxy-CTP hydrolysis is 8.5, and the optimal Mg²⁺ activation constant is 1.2mM. The experiment should be conducted at 30°C.
(2) For your safety and health, please wear a lab coat and disposable gloves during operation.
References:
[1] Fujikawa K, Kasai H. The oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP, is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein. DNA Repair (Amst). 2002 Jul 17;1(7):571-6.
| Cas No. | SDF | ||
| Formula | C9H16N3O15P3 (free acid) | M.Wt | 499.1 (free acid) |
| Solubility | Storage | Store at -20°C or below | |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
||
| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
|
1 mg | 5 mg | 10 mg |
| 1 mM | 2.0036 mL | 10.018 mL | 20.0361 mL |
| 5 mM | 400.7 μL | 2.0036 mL | 4.0072 mL |
| 10 mM | 200.4 μL | 1.0018 mL | 2.0036 mL |
Quality Control & SDS
- View current batch:
Average Rating: 5 (Based on Reviews and 30 reference(s) in Google Scholar.)