PLK1 蛋白, Human (sf9, His)

PLK1 is a serine/threonine protein kinase that centrally coordinates key functions during the M phase of the cell cycle. It regulates centrosome maturation, spindle assembly, cohesin removal, inactivates APC/C inhibitors, and controls mitotic exit and cytokinesis. PLK1 Protein, Human (sf9, His) is the recombinant human-derived PLK1 protein, expressed by Sf9 insect cells , with N-10*His labeled tag.

PLK1 是一种丝氨酸/苏氨酸蛋白激酶，在协调整个细胞周期 M 期的各种基本功能中发挥着关键作用。它调节中心体成熟和纺锤体组装，促进从染色体臂去除粘连蛋白，使后期促进复合物/环体 (APC/C) 抑制剂失活，并控制有丝分裂退出和胞质分裂。PLK1 通过结合和磷酸化已在 POLO 盒结构域识别的特定基序上磷酸化的蛋白质进行操作，从而磷酸化多种底物，包括 BORA、BUB1B/BUBR1、CCNB1、CDC25C、CEP55、ECT2、ERCC6L、FBXO5/EMI1、FOXM1 、KIF20A/MKLP2、CENPU、NEDD1、NINL、NPM1、NUDC、PKMYT1/MYT1、PRC1、RACGAP1/CYK4、SGO1、STAG2/SA2、TEX14、TOPORS、p73/TP73、TPT1、WEE1、HNRNPU 等。PLK1 的关键作用包括通过 KIZ、NEDD1 和 NINL 的磷酸化促进中心体功能和双极纺锤体组装。此外，它还通过磷酸化 CEP55、ECT2、KIF20A/MKLP2、CENPU、PRC1 和 RACGAP1 来控制有丝分裂退出和胞质分裂。PLK1 的参与延伸到着丝粒功能、姐妹染色单体凝聚力以及各种检查点蛋白的调节，使其成为控制细胞周期进展的复杂网络中的关键参与者。

Measured by its ability to catalyze 0.2 mg/mL PLKtide peptide substrate and 25 μM ATP that incubate at room temperature for 40 mins. The specific activity is >40 nmo/min/mg.

Materials
Assay buffer (5x): 200 mM Tris-HCl, pH 7.4, 100 mM MgCl₂, and 0.5 mg/mL BSA. Add fresh DTT prior to use to a final concentration of 250 μM.
PLK1 Protein, Human (sf9, His) (HY-P76550)
Substrate: PLKtide peptide substrate
Assay Kit: ADP-Glo^TM Kinase Assay
Standard: ATP/ADP

Procedure
Preparation of Standard Curve
1. Dilute the 5x assay buffer to 1x with ddH₂O.
2. Dilute ADP and ATP solutions using assay buffer (1X) to prepare 1 mL of 100 μM ADP and ATP solutions.
Well No. A1 A2 A2 A4 A5 A6 A7 A8 A9 A10 A11 A12
ADP(μL) 100 80 60 40 20 10 5 4 3 2 1 0
ATP(μL) 0 20 40 60 80 90 95 96 97 98 99 100
Add 100, 80, 60, 40, 20, 10, 5, 4, 3, 2, 1, 0 μL of 100 μM ADP and 0, 20, 40, 60, 80, 90, 95, 96, 97, 98, 99, 100 μL of 100 μM ATP solution into wells A1-A12 respectively, and mix as shown in the table above to simulate the ATP and ADP concentrations corresponding to each conversion percentage. This series constitutes the 100 μM concentration series.
3. Add 5 μL of the mixed solution to each well, followed by 5 μL of ADP-Glo^TM Reagent.
4. Incubate at room temperature for 40 min.
5. Add 10 μL of Kinase Detection Reagent to the 384-well plate.
6. Incubate at room temperature for 30 min.
7. Measure RLU values under Luminescence using a microplate reader.
8. Plot the standard curve equation with measured RLU values on the y-axis and ATP molar quantity on the x-axis.

Protein Activity Assay
1. Dilute PLK1 protein to 20 and 100 μg/mL using 1x assay buffer.
2. Prepare the mixture according to the following system:
10 mM ATP solution: 1 μL
Assay buffer (5x): 79 μL
1 mg/mL Substrate: 80 μL
3. Add 3 μL of 20 or 100 μg/mL protein solution to a 384-well plate, followed by 2 μL of the mixture. For blank wells, add 3 μL of assay buffer followed by 2 μL of the mixture.
4. Incubate at room temperature for 40 min.
5. After incubation, add 5 μL ADP-Glo^TM Reagent to each well.
6. Incubate at room temperature for 40 min.
7. After incubation, add 10 μL Kinase Detection Reagent to each well.
8. Incubate at room temperature for 30 min.
9. Measure the RLU value under Luminescence using a microplate reader.
10. CalcμLate specific activity:

Human

Sf9 insect cells

N-10*His

P53350 (M1-S603)

5347  [NCBI]

Full Length

Approximately 66-74 kDa, based on SDS-PAGE under reducing conditions, due to the glycosylation.

• 
  Greater than 95% as determined by reducing SDS-PAGE.

溶液.

Supplied as a 0.22 μm filtered solution of 50 mM Tris, 100 mM NaCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM PMSF, 25% glycerol or 50 mM PB, 300 mM NaCl, pH 7.0, 25% Glycerol, 0.1 mM EDTA, 0.5 mM PMSF, 3 mM DTT.
Note: For SPR assay, please replace the buffer. Primary amine components (e.g., Tris, imidazole) can affect protein-coupled chips.

<1 EU/μg, determined by LAL method.

N/A.

Stored at -80°C for 1 year from date of receipt. It is stable at -20°C for 3 months after opening. It is recommended to freeze aliquots at -80°C for extended storage. Avoid repeated freeze-thaw cycles.

Shipping with dry ice.