Kinase buffer, solution preparation

The deprotected oligonucleotide synthetic product is precipitated twice in ethanol, and a 0.5 fig/fd solution in water is prepared (concentration is measured from a UV absorption spectrum). One microliter of the oligo-deoxynucleotide solution is mixed with 2 fd of 10X PL, 5 fd of [y-32P]ATP (or [y-35S]ATP), 1 fd of T4 polynucleotide kinase, and 11 fd water. After incubation at 37 ° (for 45 min with [y-32P]ATP or for 2 hr with [y-35S]ATP), the reaction is stopped by the addition of 150 [A of 5 M ammonium acetate, pH 5.5, and 130 fd water and 10 fd of the yeast tRNA solution are added to the mixture before precipitation with 1 ml ethanol. After chilling at —70° for at least 15 min, the precipitate is collected by centrifugation (12,000 g, 15 min), redissolved, and submitted to two additional cycles of precipitation-redissolution. Finally, the precipitate is redissolved in 20 fd of gel loading mix and the mixture analyzed on a 8% acrylamide-7 Af urea slab gel in IX electrophoresis buffer, until the bromphenol blue has reached the middle of the gel. 

Prepare an 250-units/ml phosphorylase kinase solution by diluting the stock enzyme 1 10 with reaction buffer. 

The cyclic AMP binding reaction is conducted in a volume of 50 /xl of 50 mM sodium acetate buffer, pH 4.0. The reaction components include [ HJcyclic AMP, standard solutions or unknown samples, sufficient binding protein to bind less than 30% of cyclic AMP, and a maximally effective concentration of the protein kinase inhibitor preparation. At 0°C binding equilibrium is established within 60 min, and in the presence of the inhibitor preparation the equilibrium plateau is stable for at least 4 hr. Without the inhibitor preparation, a slow decline in bound counts occurs. 

Prepare 3 mLofacrylodan-labeled kinase solution (50-100 nM) using FLiK Buffer and place into a 4.5 mL polystyrene cuvette with a mini stir bar (see Note 16). 

Prepare a solution of 100 nM labeled kinase using the buffer prepared in step 1 and place the solution in a dispensing trough. 

Prepare the desired volume of FLiK Buffer in a Falcon tube and add 0.002-0.01% v/v Triton-XlOO to the buffer. Using the prepared FLiK Buffer, prepare a solution of 100 nM labeled kinase (see Note 48). 

Using a multichannel pipettor, add FLiK Buffer or the prepared kinase solution each to one of the HTS plates already containing inhibitor as described in step 5 of Subheading 3.2.7. 

Preparation of Radioactive FdUMP. [ H]- and [ CJFUdR are commercially available and conveniently converted to the 5 -monophosphate using thymidine kinase found in the 20-40% (NH4) 2SO4 fraction of the cell-free supernatant from Escherichia coli B. A solution (1.0 ml) containing about 1 mAf [ H]- or [ CJFUdR, 7.5 mAf ATP, 7.5 mAf MgCU, 30 mAf KF, 0.5 mg of bovine serum albumin, 70 mAf Tris-HCl (pH 7.8), and approximately 0.1 mg of the crude thymidine kinase preparation is incubated at 37°. The reaction is monitored by application of aliquotes to a micro DEAE-cellulose column (0.5 X0 8 cm) and stepwise elution with 3 ml of 5 mAf ammonium formate (pH 4.5) to elute unreacted nucleoside and 3 ml of 100 mAf of the same buffer to elute the nucleotide radioactivity is determined in each fraction to ascertain the extent of conversion. After the reaction is 80-90% complete (usually 60 min), the mixture is diluted to 20 ml with water, and purified on a DEAE-cellulose column (1X5 cm) by the method of Rustum and Schwartz- ... 

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