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dCK Phosphorylation Assay protocol

1. Buffer 5x tube 4ml to 16ml of deionized water to prepare Reaction Buffer 1x ii Transfer quantitatively the content of 2 tubes with Cofactor 1 Cofactor 2 to the tube with Reaction buffer 1x Important Do not add Cofactor 3 ATP Page 2 3 NovoCIB SAS 115 avenue Lacassagne 69003 Lyon France 69003 Lyon France K0307 01 UM V Version 150924LB contact novocib com Tel Fax 33 0 478536395 www novocib com PRECICE Enzymes amp Kits User Manual Ref K0307 01 CIB To do so iii pipet 1ml of Reaction buffer 1x to each tube with cofactors and mix them by inverting or pipeting up and down until the powder dissolved iv transfer by pipeting the content of two tubes back into a vial Reaction buffer 1x v repeat to be sure that all reagent and enzymes of the small tubes and vial are recovered Mix by gently inverting until complete dissolution Avoid bubbles vi Transfer quantitatively the content of IMPDH tube to Reaction buffer 1x with co factors vii Add 1ml of Reaction buffer 1x to Deoxyinosine tube and transfer the solution back to Reaction buffer 1x already containing Cofactor 1 Cofactor 2 and IMPDH viii Solubilize the content of Human dCK enzyme tube by adding 1ml of complete Reaction buffer 1x with co factors and IMPDH transfer by pipeting the content of the tube back into a vial Reaction buffer 1x Composition of complete reaction
2. enzyme Add 250uL of deionized water to IMPDH tube Agitate gently until complete dissolution of the powder V 2 Preparation of standard reaction buffer 1x i Add the content of Reaction Buffer 5x tube 4ml to 16ml of deionized water to prepare Reaction Buffer 1x ii Transfer quantitatively the content of 2 tubes with Cofactor 1 and Cofactor 2 to the tube with Reaction buffer 1x To do so iii Add 1ml of Reaction buffer 1x to each tube with cofactors and mix them by inverting or pipeting up and down until the powder dissolved iv transfer by pipetting the content of two tubes back into a vial Reaction buffer 1x v repeat to be sure that all reagent and enzymes of the small tubes and vial are recovered Mix by gently inverting until complete dissolution Avoid bubbles vi Transfer quantitatively the content of IMPDH tube to Reaction buffer 1x with co factors vii Add 200uL of deionized water to Cofactor 3 tube ATP Agitate until dissolved Take off 20uL of thus prepared ATP solution to Reaction buffer 1x already containing Cofactor 1 Cofactor 2 and IMPDH viii Solubilize the content of Human dCK enzyme tube by adding 1m of complete Reaction buffer 1x with co factors and IMPDH transfer by pipeting the content of the tube back into a vial Reaction buffer 1x Composition of complete reaction buffer 100MM Tris HCI 250mM KCI 10mM MgCl2 BSA 0 5mg ml
3. ribo and Figure 1 deoxyribonucleosides and ribavirine Nucleosides and nucleoside analogues 2uL of 100mM solution were added to 0 75 96 well plate followed by the addition 0 7 of 200uL of standard reaction mix containing DTT NAD IMPDH and dCK 10 min later the reaction was started gs trol by the addition of ATP solution 10uL COMTO per well and the increase in deoxyguanosine mM absorbance at 340nm was followed for uridine 1mM 30min deoxyuridine 1mM thymidine 1mM gemcitabine 0 5mM cytidine 1mM deoxycytidine 1mM Results ribavirine Of 8 deoxy and ribonucleosides tested only deoxycytidine and gemcitabine competitively inhibit deoxyinosine phosphorylation by dCK which totally consistent with previously published data on their 0 5 10 15 20 25 30 35 substrate properties Time after ATP addition second incubation min Page 3 3 NovoCIB SAS 115 avenue Lacassagne 69003 Lyon France 69003 Lyon France K0307 01 UM V Version 150924LB contact novocib com Tel Fax 33 0 478536395 www novocib com
4. 5mM DTT 5mM NAD 250uM ATP IMPDH 50mU ml human recombinant dCK 5mU ml V 3 Preparation of deoxyinosine solution for starting the reaction Add 960ul of deionized water to Deoxyinosine tube and mix by inverting 1ml of 20mM dIR solution is obtained VI Following dCK activity in vitro VI 1 Pre incubation phase 15 i Program the plate reader in a kinetics mode with the measurements done every 1 minutes absorbance at 340 nm 37 C agitation before the kinetics for 1 min duration time 15min ii Add 200uL of standard reaction buffer per well iii Agitate and measure absorbance at 340nm Agqo Record this first set of data 5 2 2 Start the reaction and incubate 40 i Eject the plate from the plate reader ii Program the plate reader in a kinetics mode with the measurements done every 1 minutes absorbance at 340 nm 37 C agitation before the kinetics for 1 min duration time 30 40min iii Start the reaction by adding 10uL of 20mM of Deoxyinosine per well mM dIR final concentration iv Place the plate in the plate reader and start the measurements Record second set of data Vil Experimental Protocol Il for nucleoside analogues dCK inhibitors Important Spin all tubes before solubilizing VII 1 Reconstitute IMPDH enzyme Add 250uL of deionized water to IMPDH tube Agitate gently until complete dissolution of the powder VII 2 Preparation of standard reaction buffer 1x i Add the content of Reaction
5. PRECICE Enzymes amp Kits User Manual Ref K0307 01 CIB PRECICE dCK Screening Assay Kit For research use only Not for use in diagnostic procedures Introduction dCK Assay Kit was specially designed to follow the enzymatic activity of purified deoxycytidine kinase dCK EC 2 7 1 74 in vitro This user manual gives the instructions for standard assays in 96 well plate The principle of the assay is based on the use of deoxyinosine dIR as a substrate of dCK and a coupled reaction involving a highly active IMPDH Inosine Monophosphate Dehydrogenase bacterial recombinant for a direct measurement of the deoxyinosine monophosphate dIMP formed by dCK 1 Inthe presence of dIR and ATP dCK catalyses the phosphorylation of dIR to form dIMP and ADP N ATP ADP N N e O Nf i OL N a N dCK OH R N amp HO HO Deoxyinosine dIR Deoxyinosine monophosphate dIMP 2 The dIMP formed is then oxidized to deoxyxanthosine monophosphate dXMP by IMPDH in the presence of NAD leading to NADH formation NADH N HO P O OH ane IMPDH bey HO N HO O Deoxyinosine monophosphate dIMP Deoxyxanthosine monophosphate dXMP This coupling reaction is immediate when IMPDH activity is much higher than dCK activity in the assay The enzymatic activity of dCK which corresponds to the formation kinetics of dIMP is then stoichiometrically and directly monitored by the formation kinetics of NADH The velocity of NADH2 for
6. buffer 100MM Tris HCI 250mM KCI 10mM MgCl2 BSA 0 5mg ml 5mM DTT 5mM NAD 1mM deoxyinosine IMPDH 50mU ml human recombinant dCK 5mU ml VII 3 Preparation of ATP solution for starting the reaction Add 1ml of deionized water to the tube Cofactor 3 containing 30mg of ATP powder Mix until dissolved 50mM ATP solution is obtained VIII Following dCK activity in vitro VI 1 Pre incubation phase 15 i Program the plate reader in a kinetics mode with measurements done every 1 minutes absorbance at 340 nm 37 C agitation before the kinetics for 1 min duration time 15min ii Add 200uL of standard reaction buffer per well iii Agitate and measure absorbance at 340nm A340 Record this first set of data 5 2 2 Start the reaction and incubate 40 i Eject the plate from the plate reader ii Program the plate reader in a kinetics mode with shaking for 1min and the measurements done every 1 minutes absorbance at 340 nm 37 C agitation before the kinetics for 1 min duration time 60min iii Start the reaction by adding 10uL of 50mM ATP per well 2 5mM ATP final concentration iv Place the plate in the plate reader and start the measurements Record second set of data Assay validation Absorbance at 340 Specific competitive inhibition of deoxyinosine phosphorylation by dCK 0 65 0 6 5 0 55 0 5 5 0 45 0 4 5 0 35 0 3 0 25 T T T T T T d by deoxycytidine and gemcitabine in comparison to other
7. mation is measured with a spectrophotometer at 340nm molar extinction coefficient of NADH2 at 340nm 6220 M cm ll Kit Contents A standard PRECICE dCK Screening Assay Kit one 96 well plate contains one tube IMPDH one tube Cofactor 1 one tube Cofactor 2 one tube Cofactor 3 one vial Reaction Buffer 5x one tube Deoxyinosine one tube Human dCK enzyme 100mU 8 Transparent 96 well plate round bottom 96 well plate Corning Costar ref 3797 PO OT oN lll Equipments required 1 Plate agitator 2 Plate reader fitted with a filter 340nm IV Storage PRECICE dCK Screening Assay Kit must be stored at 20 C until used IMPORTANT The following instructions are given to measure the activity of CK enzyme in vitro in a range allowing this measurement by spectrophotometry as described here below NovoClIB does not guarantee the use of its PRECICE dCK Screening Assay Kit or of one or several of its components in other conditions than those described in this user manual and or for other purpose than R amp D Page 1 3 NovoCIB SAS 115 avenue Lacassagne 69003 Lyon France 69003 Lyon France K0307 01 UM v Version 150924LB contact novocib com Tel Fax 33 0 478536395 www novocib com PRECICE Enzymes amp Kits CIB User Manual Ref K0307 01 V Experimental Protocol for non nucleoside dCK inhibitors Important Spin all tubes before solubilizing V 1 Reconstitute IMPDH

dCK Phosphorylation Assay protocol

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