• 
dCK Inhibition - Contract Research Service for In vitro Screening
Our Contract Research Service for dCK Inhibition screening is performed on Human recombinant Deoxycytidine Kinase (Ref. E-Nov3), produced and purified by NOVOCIB.
Our Contract Research Service for dCK Inhibition screening is performed on Human recombinant Deoxycytidine Kinase (Ref. E-Nov3), produced and purified by NOVOCIB.
Aim: Screening of compounds for their abilities to inhibit human Deoxycytidine Kinase (dCK) in vitro. Determination of the inhibition kinetics of a given compound on human recombinant dCK and measurement of its IC50 value.
Human dCK: The dCK enzyme used in the assays is a human recombinant Deoxycytidine Kinase, cloned by NOVOCIB from human cells, expressed in E. coli, and produced and purified by NOVOCIB.
Enzyme QC: The dCK enzyme purity is controlled before every assay by SDS-PAGE. Enzyme activity is systematically controlled before performing any assay.
Replicate assays: One point is defined as a well per compound and per concentration tested. dCK in vitro assays are usually performed in duplicate (2 wells per compound and per concentration). Triplicates are available upon request.
dCK inhibition control: Any validated dCK inhibitor, when provided by our client, can be used as positive control for dCK inhibition. Both negative and positive controls are done in duplicate.
Enzymatic Reaction: In vitro screening for dCK inhibition is based on a spectrophotometric measurement of Deoxycytidine Kinase activity using a coupling reaction with IMPDH. For further details, please refer to our "dCK Assay Kit".
The assays are performed on 96-well microplate, at 37°C, in 200µl of reaction buffer (Tris-HCl 0.1M, pH7.9, KCl 0.1M, Mg Acetate 12mM, ATP 2mM, NAD 2mM, DTT 5mM, IMPDH 25mU/ml, Deoxyinosine 5mM).
Procedure: Every assay, from one to 90 points, is done with one negative control, containing DMSO with no inhibitor, and:
- For Screening Assays: 2 positive controls containing a validated dCK inhibitor, when provided, at two different final concentrations (usually, IC50 and a highest concentration leading to 100% inhibition.
- For Inhibition Kinetics (IC50): 8 or 12 positive controls containing a validated dCK inhibitor, when provided, at different concentrations, equally spaced by 3- or 2-fold dilutions respectively, to cover a 3.3-log wide range.
Controls are done in duplicate. If an additional microplate is needed, it includes the complete set of controls (in duplicate). Additional concentrations of inhibitor can be tested.
In case of positive assays, controls of IMPDH non-inhibition are done in parallel with IMPDH inhibition by mycophenolic acid (MPA), according our procedure for IMPDH Inhibition Screening.
Ask for Quotation