PLK1 Protein, 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, a serine/threonine-protein kinase, plays a pivotal role in orchestrating various essential functions throughout the M phase of the cell cycle. It regulates centrosome maturation and spindle assembly, facilitates the removal of cohesins from chromosome arms, inactivates anaphase-promoting complex/cyclosome (APC/C) inhibitors, and governs mitotic exit and cytokinesis. Operating by binding and phosphorylating proteins that are already phosphorylated on specific motifs recognized by the POLO box domains, PLK1 phosphorylates an extensive array of substrates, including 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, and others. PLK1's crucial roles include promoting centrosome functions and bipolar spindle assembly through the phosphorylation of KIZ, NEDD1, and NINL. Additionally, it governs mitotic exit and cytokinesis by phosphorylating CEP55, ECT2, KIF20A/MKLP2, CENPU, PRC1, and RACGAP1. PLK1's involvement extends to kinetochore functions, sister chromatid cohesion, and the regulation of various checkpoint proteins, positioning it as a key player in the intricate network governing cell cycle progression.

The specific activity was determined to be > 3 nmol/min/mg using casein as substrate.

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 kDa

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

Solution

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.