Recombinant human CK1 epsilon protein (ab104024) (ab104024)

Please see the requested protocol below. Please contact me again if you have any further questions.

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Kinase assay protocol for CK1-epsilon, Abcam ab104024

Thaw [32P]-ATP assay cocktail in a designated radioactive working area.Thaw the Active CK1-epsilon, Kinase Assay Buffer, Substrate and Kinase Dilution Buffer on ice.





In a pre-cooled microfuge tube, add the following reaction components, bringing the initial reaction volume up to 20μl.





Component 1. 10μl of diluted active CK1-epsilon

Component 2. 5μl of 1mg/ml stock solution of substrate.

Component 3. 5ul distilled water. (4oC)



Set up blank control as outlined in step 3, excluding the addition of substrate. Replace the substrate with an equal volume of distilled water.
Initiate the reaction by the addition of 5ul [32P]-ATP Assay Cocktail bringing the final volume up to 25μl and incubate the mixture in a water bath at 30oC for 15 minutes.
After the 15 minute incubation period, terminate the reaction spotting 20ul of the reaction mixture onto individual pre-cut strips of phosphor cellulose P81 paper.
Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (dilute 10ml of phosphoric acid and make a 1 liter solution with dH2O) with constant gentle stirring. It is recommended that the strip should be washed a total of 3 intervals for approximately 10 minutes each.
Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
Determine the corrected cpm by removing the blank control value (see step 4) for each sample and calculate the kinase specific activity as below:



Calculation of [P33]-ATP Specific Activity (SA) (cpm/pmol)

Specific activity (SA) = cpm for 5 μl [33P]-ATP / pmoles of ATP (in 5 μl of a 250 μM ATP stock solution, i.e., 1250 pmoles)

Kinase Specific Activity (SA) (pmol/min/μg or nmol/min/mg)

Kinase specific activity (pmol/ min/ ug) = Corrected cpm from reaction / (Specific activity of [32P]-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in ug) x [(Reaction volume / spot volume)]






Active Kinase
Active CK1-epsilon (0.1μg/μl) diluted with Kinase Dilution Buffer. Assay as outlined in sample activity plot. (Note: these are suggested working dilutions and it is recommended that the researcher perform a serial dilution of Active CK1-epsilon for optimal results).

[32P]-ATP Assay Cocktail
Prepare 250μM [33P]-ATP Assay Cocktail in a designated radioactive working area by adding the following components: 150μl of 10mM ATP Stock Solution, 100 μl [32P]-ATP (1mCi/100μl), 5.75ml of Kinase Assay Buffer I. Store 1ml aliquots at –20oC.

Kinase Dilution Buffer
Kinase Assay Buffer I diluted at a 1:4 ratio (5X dilution) with 50ng/μl BSA and 5% glycerol solution.


10mM ATP Stock Solution
Prepare ATP stock solution by dissolving 55mg of ATP in 10ml of Kinase Assay Buffer I. Store 200μl aliquots at –20oC.

Kinase Assay Buffer I
Buffer components: 25mM MOPS, pH 7. 2, 12.5mM β-glycerol-phosphate, 25mM MgC12, 5mM EGTA, 2mM EDTA. Add 0.25mM DTT to Kinase Assay Buffer prior to use.

Substrate
Casein, dephosphorylated, a protein substrate, was diluted in distilled water to a final concentration of 1mg/ml.