Recombinant Human Active PLK1 Protein, CF

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The approximate molecular weight is 70 kDa and the average purity is 73%.

Product Details

Summary
Reactivity HuSpecies Glossary
Applications Bioactivity
Format
Carrier-Free

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Recombinant Human Active PLK1 Protein, CF Summary

Details of Functionality
The activity of PLK1 is 14-20 nmol/min/mg using a dephospho-casein substrate (see Activity Assay Protocol).
Source
Spodoptera frugiperda, Sf 9 (baculovirus)-derived human PLK1 protein
Accession # NM_005030
Accession #
N-terminal Sequence
Using an N-terminal His tag
Protein/Peptide Type
Recombinant Proteins
Gene
PLK1
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane

Applications/Dilutions

SDS-PAGE
70 kDa
Publications
Read Publication using
3804-KS in the following applications:

Packaging, Storage & Formulations

Storage
This product is stable at ≤ ‑70° C for up to 1 year from the date of receipt. For optimal storage, aliquot into smaller quantities after centrifugation and store at recommended temperature. Avoid repeated freeze-thaw cycles.
Buffer
Supplied in 50 mM sodium phosphate (pH 7.0), 300 mM NaCl, 0.25 mM DTT, 150 mM imidazole, 0.1 mM PMSF, and 25% glycerol.
Purity
>70%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
Assay Procedure
  • Active Kinase - Active PLK1 (0.1 μg/μL) diluted with Kinase Dilution Buffer. Note: These are suggested working dilutions. Optimal dilutions should be determined by each laboratory for each application.
  • Kinase Assay Buffer, pH 7.2 - 25 mM MOPS, 12.5 mM beta -glycerolphosphate, 25 mM MgCl2, 5 mM EGTA, 2 mM EDTA. Add 0.25 mM DTT to the Kinase Assay Buffer prior to use.
  • Kinase Dilution Buffer, pH 7.2 - Kinase Assay Buffer diluted 5-fold with 50 ng/μL BSA solution.
  • 10 mM ATP Stock Solution - Prepare the ATP Stock Solution by dissolving 55 mg of ATP in 10 mL of Kinase Assay Buffer. Store 200 μL aliquots at ≤ -20° C.
  • [33P]-ATP Assay Cocktail - Prepare 250 μM [33P]-ATP Assay Cocktail in a designated radioactive work area by combining 150 μL of 10 mM ATP Stock Solution, 100 μL of [33P]-ATP (1 mCi/100 μL), and 5.75 mL of Kinase Assay Buffer. Store 1 mL aliquots at ≤ -20 °C.
  • Substrate - Dephospho-Casein diluted in distilled or deionized water to a final concentration of 1 mg/mL.
  1. Thaw the [33P]-ATP Assay Cocktail in a shielded container in a designated radioactive work area.
  2. Thaw the Active PLK1, Kinase Assay Buffer, Substrate, and Kinase Dilution Buffer on ice.
  3. In a pre-cooled microfuge tube, add the following reaction components bringing the initial reaction volume up to 20 μL.
    a. Diluted Active PLK1 10 μL
    b. Substrate (1 mg/mL; on ice): 5 μL
    c. Distilled or deionized water (on ice): 5 μL
  4. Set up the blank control as outlined in step 3, excluding the addition of the substrate. Replace the substrate with an equal volume of distilled or deionized water.
  5. Initiate the reaction with the addition of 5 μL [33P]-ATP Assay Cocktail, bringing the final volume up to 25 μL. Incubate the mixture in a water bath at 30 °C for 15 minutes.
  6. After the 15 minute incubation, terminate the reaction by spotting 20 μL of the reaction mixture onto individual pre-cut strips of phosphocellulose P81 paper.
  7. Air dry the pre-cut P81 strip and sequentially wash in a 1% phosphoric acid solution (add 10 mL of phosphoric acid to 990 mL of distilled or deionized water) with constant gentle stirring. It is recommended that the strips be washed a total of three times for approximately 10 minutes each.
  8. Count the radioactivity on the P81 paper in the presence of scintillation fluid in a scintillation counter.
  9. Determine the corrected cpm by subtracting the blank control value (see step 4) for each sample and calculate the kinase specific activity as outlined below:


    Calculation of [33P]-ATP Specific Activity (SA) (cpm/pmol)
    Specific Activity (SA) = cpm for 5 μL [33P]-ATP/pmol of ATP (in 5 μL of a 250 μM ATP stock solution; i.e. 1250 pmol)

    Calculation of Kinase Specific Activity (SA) (pmol/minutes/μg or nmol/minutes/mg)
    Corrected cpm from reaction / [(SA of 33P-ATP in cpm/pmol) x (Reaction time in minutes) x (Enzyme amount in μg or mg)] x [(Reaction volume) / (Spot Volume)]

Notes

This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant Human Active PLK1 Protein, CF

  • cell cycle regulated protein kinase
  • EC 2.7.11
  • EC 2.7.11.21
  • PLK
  • PLK1
  • PLK-1
  • polo (Drosophia)-like kinase
  • polo like kinase
  • polo-like kinase 1polo-like kinase (Drosophila)
  • Serine/threonine-protein kinase 13
  • serine/threonine-protein kinase PLK1
  • STPK13

Background

PLK1 is a member of the Polo-Like Kinase family that localizes to centrosomes or spindle pole bodies and undergoes dramatic subcellular relocation during the cell cycle. Deregulated activities of PLKs often result in abnormalities in centrosome duplication, maturation, and/or microtubule dynamics (1). PLKs also regulate the function of the Golgi complex. Deregulated expression of human PLK1 is strongly correlated with the development of many types of malignancies, and ectopic expression of PLK1 dominant negative protein leads to rapid cell death (2).

  1. Nigg, E.A. et al. (1996) Exp. Cell Res. 229:174.
  2. Dai, W. et al. (2003) Prog. Cell Cycle Res. 5:327.

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Publications for PLK1 (3804-KS)(1)

We have publications tested in 1 confirmed species: N/A.

We have publications tested in 1 application: Bioassay.


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Bioinformatics

Gene Symbol PLK1
Uniprot