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Peer-reviewed

In Gel Kinase Assay

凝胶内激酶测定法

Gaston A. Pizzio Gaston A. Pizzio
Pedro L. Rodriguez Pedro L. Rodriguez

发布: 2017年03月05日第7卷第5期 DOI: 10.21769/BioProtoc.2170 浏览次数: 15173

评审: Rainer MelzerHarrie van ErpAnonymous reviewer(s)

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Abstract

Proper spatiotemporal regulation of protein phosphorylation in cells and tissues is required for normal development and homeostasis. We present the protocol ‘In Gel Kinase Assay’, which is useful for protein kinase activity measurements from crude protein extracts. We have successfully used ‘In Gel Kinase Assay’ protocol to show that the Arabidopsis thaliana sextuple mutant in the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors; line pyr/pyl112458) is impaired in ABA-mediated activation of SnRK2.2, SnRK2.3 and OST1/SnRK2.6, as much as the triple mutant snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012).

Keywords: Kinases (激酶) search Phosphorylation (磷酸化) search in vivo (体内) search Enzymatic activity (酶活性) search SnRKs (SnRKs) search ABA (脱落酸) search Arabidopsis (拟南芥) search

Background

The phytohormone abscisic acid (ABA) is a key signal involved in plant growth and development as well as in plant response to abiotic and biotic stress. The ABA perception and signaling pathway is composed of PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR-ABA receptors), PP2Cs phosphatases and SnRK2s kinases (reviewed in Antoni et al., 2011). The module receptor-ABA-phosphatase controls the phosphorylation signaling cascades in a ligand-dependent manner through regulation of ABA-activated SnRK2s. In turn, SnRK2s kinases phosphorylate a myriad of effectors, both in the nucleus and in the cytoplasm, from transcription factors (e.g., ABFs) to ion channels (e.g., SLAC1). We show here the protocol ‘In Gel Kinase Assay’ in details. This protocol was developed for protein kinase activity measurement in plant tissues protein extracts as well as from purified recombinant kinases. In brief: an SDS-polyacrylamide gel is prepared containing the ∆C-ABF2 peptide (a specific SnRK2.2, SnRK2.3, OST1/SnRK2.6 kinase substrate). ∆C-ABF2 peptide is trapped in the SDS-polyacrylamide gel mesh and it does not migrate during electrophoresis. On the other hand, SDS-treated protein samples, without boiling and without any reducing agent (e.g., DTT or B-ME), are only partially denatured and can be re-naturalized in some degree after washing SDS out. Moreover, kinase activity at a high sensitive level can be measured using [Gamma-32p]ATP. In this way, together with the kinase activity value we also have the electrophoretic mobility value associated with the kinase activity. Using a plant crude protein extract, and after sample electrophoresis and in gel protein renaturalization step, we can measure specific SnRK2.2/2.3/2.6 (SnRK2s) kinase activity in gel. We have applied this protocol to characterize SnRK2s activity in a PYR/PYL/RCAR-ABA receptor sextuple mutant. We show that the sextuple mutant line pyr/pyl112458 is impaired in ABA-mediated activation of SnRK2s, as much as the triple mutant snrk2.2/2.3/2.6 in Arabidopsis thaliana (result published in Gonzalez-Guzman et al., 2012).

Materials and Reagents

  1. Eppendorf and Falcon tubes (1.5 ml and 50 ml respectively; generic)
  2. Pipettes and tips (generic)
  3. Micropore tape (3M, catalog number: 1530-0 )
  4. Sterile round Petri dishes (100 mm diameter x 20 mm height; generic: e.g., Greiner Bio One, catalog number: 664161 )
  5. Extra thick blot filter paper, 7.5 x 10 cm (Bio-Rad Laboratories, catalog number: 1703965 )
  6. Arabidopsis thaliana seeds: Col-0 (NASC, N1093), pyr/pyl112458 and snrk2.2/2.3/2.6 (Gonzalez-Guzman et al., 2012)
  7. Optional: purified recombinant kinase OST1 (SnRK2.6-6his; homemade: Vlad et al., 2009 and 2012)
  8. Sterile deionized water (generic)
  9. Liquid N2 (generic)
  10. Ice (generic)
  11. ABA (+cis, trans-Abscisic acid) (BIOSYNTH, catalog number: A-0120 )
  12. Bio-Rad Protein Assay Kit I (Bio-Rad Laboratories, catalog number: 5000001 )
  13. Bio-SafeTM Coomassie Stain reagent (Bio-Rad Laboratories, catalog number: 1610786 )
  14. ∆C-ABF2 peptide (amino acids 1 to 173; 18.4 KD; homemade: Dupeux et al., 2011; Antoni et al., 2012)
    Note: Commercially available alternative MBP (myelin basic protein) (Sigma-Aldrich, catalog number: M1891 ).
  15. SDS-PAGE reagents (listed in He, 2011)
  16. Ethanol (generic)
  17. Polyethylene glycol sorbitan monolaurate (Tween-20) (Sigma-Aldrich, catalog number: P1379 )
  18. Murashige and Skoog medium (MS) (PhytoTechnology Laboratories®, catalog number: M524 )
  19. 2-N-morpholino-ethane-sulfonic acid (MES) (Sigma-Aldrich, catalog number: M8250 )
  20. Sucrose (VWR, BDH®, catalog number: BDH0308 )
  21. Agar-agar (Sigma-Aldrich, catalog number: A1296 )
  22. Potassium hydroxide (KOH) (Fisher Scientific, catalog number: P250 )
  23. 2-Amino-2-(hydroxymethyl)-1,3-propanediol (Tris-base) (Sigma-Aldrich, catalog number: 252859 )
  24. HCl
  25. 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid) (HEPES) (Sigma-Aldrich, catalog number: H3375 )
  26. DL-dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815 )
  27. EDTA (Sigma-Aldrich, catalog number: E5134 )
  28. EGTA (Sigma-Aldrich, catalog number: E3889 )
  29. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S7920 )
  30. Sodium orthovanadate (Na3VO4) (Sigma-Aldrich, catalog number: 450243 )
  31. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  32. Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: M8266 )
  33. Adenosine 5’-triphosphate disodium salt hydrate (ATP) (Sigma-Aldrich, catalog number: A1852 )
  34. Glycerol 2-phosphate disodium salt hydrate (BGP) (Sigma-Aldrich, catalog number: G9422 )
  35. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  36. Protease inhibitor cocktail (Sigma-Aldrich, catalog number: S8830 )
  37. Sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771 )
  38. 2-(5-Bromo-2-pyridylazo)-5-(diethylamino) (Bromo-phenol-blue) (Sigma-Aldrich, catalog number: 180017 )
  39. Polyethylene glycol tert-octylphenyl ether (Triton X-100) (Sigma-Aldrich, catalog number: T8787 )
  40. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: 05470 )
  41. [Gamma-32p]ATP, 10 μCi/ml - 3,000 Ci/mmol (PerkinElmer, catalog number: BLU502A )
  42. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: T6399 )
  43. Sodium pyrophosphate (Na2PPi) (Sigma-Aldrich, catalog number: 71501 )
  44. Seed sterilization solution (see Recipes)
  45. Solid MS (see Recipes)
  46. Stock reagent (see Recipes)
    1. 1 M Tris-HCl (pH 6.8 and 7.5)
    2. 1 M HEPES-KOH (pH 7.5)
    3. 1 M DTT
    4. 0.5 M EGTA
    5. 0.5 M EDTA
    6. 1 M NaF
    7. 100 mM Na3VO4
    8. 100 mM PMSF
    9. 1 M MgCl2
    10. 100 mM ATP
  47. Protein extraction buffer (see Recipes)
  48. 2x Laemli buffer (see Recipes)
  49. Washing buffer (see Recipes)
  50. Protein renaturalization buffer (see Recipes)
  51. Cold reaction kinase buffer (see Recipes)
  52. Hot reaction kinase buffer (see Recipes)
  53. STOP-washing buffer (see Recipes)

Equipment

  1. Radiation safety gears and personal protection (generic)
  2. Pipette  
  3. Flow hood (generic: e.g., CLEATECH, catalog number: 1000-11-E )
  4. Plant growth chamber (generic: e.g., BioChambers, model: FXC-9 )
  5. Benchtop microcentrifuge (generic: e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: SorvallTM LegendTM Micro 17 , catalog number: 75002430)
  6. SDS-PAGE system (Bio-Rad Laboratories, model: Mini-PROTEAN® Tetra Vertical Electrophoresis Cell )
  7. Vacuum gel dryer (generic: e.g., Bio-Rad Laboratories, model: 583 Gel Dryers )
  8. Phosphorimager screen and cassette (FUJIFILM)
  9. Phosphorimager (FUJIFILM, model: FLA-5100 )
  10. Autoclave (generic)
  11. Mortar (100 mm diameter x 60 mm height; generic)
  12. Spatula and forceps (generic)
  13. Plastic incubation box (for Polyacrylamide gel incubation and washing; generic)
  14. Stirring bars (50 mm long; generic)
  15. Timer (generic)
  16. Rotary shaker (generic)
  17. Fridge (4 °C; generic)
  18. Freezer (-20 °C and -80 °C; generic)
  19. Magnetic stirrer (generic)
  20. pH meter (generic: e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: Orion StarTM A111 )
  21. Balance (generic: e.g., Sartorius, model: Cubis® Precision Balance )
  22. Vortex (generic)
  23. Spectrophotometer (generic: e.g., Thermo Fisher Scientific, Thermo ScientificTM, model: EvolutionTM 300 )

Software

  1. ImageJ (optional)

Procedure

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

  1. Pizzio, G. A. and Rodruiguez, P. L. (2017). In Gel Kinase Assay. Bio-protocol 7(5): e2170. DOI: 10.21769/BioProtoc.2170.
  2. Gonzalez-Guzman, M., Pizzio, G. A., Antoni, R., Vera-Sirera, F., Merilo, E., Bassel, G. W., Fernandez, M. A., Holdsworth, M. J., Perez-Amador, M. A., Kollist, H. and Rodriguez, P. L. (2012). Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid. Plant Cell 24(6): 2483-2496.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 蛋白质

生物化学 > 蛋白质 > 活性

生物化学 > 蛋白质 > 电泳

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