Assay protocol

Principle: The PLK1 assay is performed using the ADP-GloTM Kinase Assay kit which quantifies the amount of ADP produced by the PLK1 reaction. The ADP-GloTM Reagent is added to terminate the kinase reaction and to deplete the remaining ATP, and then the Kinase Detection Reagent is added to convert ADP to ATP and to measure the newly synthesized ATP using luciferase/luciferin reaction.

Materials

1.Kinase assay buffer(5X): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl2 and 0.5 mg/mL BSA, 250 μM DTT

2.Kinase assay buffer(1X): 40 mM Tris-HCl, pH 7.4, 20 mM MgCl2, 0.1 mg/mL BSA, 50 μM DTT

3.PLK1 GST Tag & His Tag Protein, Human

4.ADP-Glo Kinase Assay (UA, Catalog # UA070101)

5.Substrate: PLKtide peptide (Sinobiological, Catalog # P41-58)

6.Solid white multi-well plate (384-well plate) (Corning, Catalog #3572)

7.Plate Reader (PerkinElmer)

Produce

1.Prepare a substrate/ATP mixture as follows (25 μM example).

Sample Name	Amount (μL)
10 mM ATP Solution	1
Kinase Assay Buffer III (5x)	79
Substrate at 1 mg/mL	80

2. Dilute the PLK1 to 20 µg/mL, 10 µg/mL and 5 µg/mL in Kinase Assay Buffer (1x) and dispense 3 μL into each well of a 384-well plate.

3. Initiate the reaction by adding 2 μL of the detection system prepared in Step 1 to each well. Include a detection system with 3 μL Kinase Assay Buffer (1x) as Blank. The reaction volume is 5 μL.

4. Incubate the reaction at room temperature (22–25℃) for 40 minutes.

5.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25 ℃).

6.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25 ℃).

7.Read at luminescence, respectively in endpoint mode.

8.Calculate specific activity.

• Standard Curve

1.Dilute the ATP and ADP to 25 μM in Kinase Assay Buffer (1x).

2.Mix 25 μΜ ATP and 25 μM ADP to form an ATP+ADP solution provided below and dispense 5 μL into each well of a 384-well plate.

Well Number	1	2	3	4	5	6	7	8	9	10	11	12
25μM ADP (μL)	100	80	60	40	20	10	5	4	3	2	1	0
25μM ATP (μL)	0	20	40	60	80	90	95	96	97	98	99	100

3.Add 5 μL of ADP-Glo Reagent to the completed reaction, mix briefly and incubate for 40 minutes at room temperature (22–25 ℃).

4.Add 10 μL of Detection Reagent and incubate the plate for 30 minutes at room temperature (22–25 ℃).

5.Read at luminescence, respectively in endpoint mode.

6.Detect optical signals and establish conversion curves.

Specific Activity (pmol/min/μg) =	ATP (pmol)-Blank
	Incubation time(min) ×amount of enzyme (μg)

PLK1 GST&His Tag Protein, Human