In Gel Kinase Assay
凝胶内激酶测定法   

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The Plant Cell
Jun 2012

 

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), Phosphorylation (磷酸化), in vivo (体内), Enzymatic activity (酶活性), SnRKs (SnRKs), ABA (脱落酸), Arabidopsis (拟南芥)

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

Note: Although this protocol was developed for plant tissue protein extracts as well as recombinant proteins, it potentially can also be applied for protein extracts from any other source. Even more, this protocol could also be valid for samples obtained from immunoprecipitation assay (Vlad et al., 2009).

  1. Seed sterilization and plant growth
    1. Put 5 mg (200-300 seeds) of Arabidopsis seeds into an Eppendorf tube, add 750 μl sterilization solution (Recipe 1) and shake for 20 min at room temperature.
      Note: In this protocol we tested: pyr/pyl sextuple mutant line (pyr/pyl112458), snrk triple mutant line (snrk2.2/2.3/2.6) and Col-0 as a control line (described in Gonzalez-Guzman et al., 2012).
    2. Remove the sterilization solution (by pipetting with sterile tips) in flow hood and wash seeds with 750 μl sterile distilled water (shaking it for 15 min at room temperature). Repeat this step 3 times.
    3. Stratify seeds in 750 μl fresh sterile distilled water at 4 °C for 3 days in the dark.
    4. Sow seeds on sterile solid MS plates (Recipe 2).
      Note: Keep a density of approximately one seed per cm2 (crowded plates produce unhealthy seedlings).
    5. Close and seal plates with micropore tape.
      Note: Use only one layer of micropore tape. More than one layer prevents both: gaseous exchange and healthy plant growth.
    6. Incubate plates for 12 days in a controlled environment growth chamber at 22 °C under a 16-h light/8-h dark photoperiod at 100 µEm-2 sec-1.
      Note: Time and growth condition could be variable depending on experimental requirements.

  2. Total protein extraction and SDS-PAGE
    1. Harvest seedlings from plates (500 mg) and freeze it in liquid N2. Store at -80 °C until use.
      Note: In our experimental design 100 µM ABA or mock treatment (sprayed - 30 min) was required before freezing.
    2. Grind plant tissues into fine powder using mortar and liquid N2.
    3. Transfer 400 mg of the fine powder into a 2 ml Eppendorf tube using a cooled (N2) spatula; add 1 ml of cold extraction buffer (Recipe 3) and mix well by vortexing for 15 sec.
      Note: Keep tube on ice until centrifugation (proceed with the next sample).
    4. Centrifuge for 15 min at 4 °C (maximum speed, 14,000 x g).
    5. Transfer supernatant into a new 1.5 ml Eppendorf tube and store at -80 °C until use.
      Note: Before storing tubes at -80 °C, take a sample of each one (50 µl) for protein quantification. Repeated sample freeze and thaw can decrease enzymatic activity.
    6. Proceed with protein quantification.
      Note: Any protein quantification method should be fine. We choose Bradford Protein Assay from Bio-Rad (follow manufacturers’ protocol)
    7. Prepare 10% polyacrylamide gel (for SDS-PAGE) and include before polymerization 800 µg of ∆C-ABF2 peptide (in the resolving gel).
      Notes:
      1. We choose miniprotean III SDS-PAGE system from Bio-Rad and a gel of 1.5 mm width.
      2. We use as specific SnRK2.2/2.3/.2.6 kinase substrate the His6-∆C-ABF2 peptide (amino acids 1 to 173; 18.4 KD). ∆C-ABF2 cloning and recombinant protein preparation was described in: Dupeux et al., 2011 and Antoni et al., 2012 (see a brief description of these methods in Notes section).
      3. Potentially other recombinant kinase substrates could be used depending on which kinases will be assayed. Even synthetic or commercially available peptides, i.e., MBP (myelin basic protein), should be suitable.
      4. For SDS-PAGE preparation follow the BIOPROTOCOL e80 (He, 2011)
    8. Prepare loading samples: mix 40 µg of total protein plant extract (obtained in step B5 and thawed on ice) with 50 µl 2x Laemli buffer (Recipe 5) and add sterile deionized H2O to reach 100 µl final volume.
      Notes:
      1. Do not boil the samples. Boiling produces irreversible protein denaturation and loss of enzymatic activity. (Important)
      2. Quantity of total protein extract could be variable depending on kinase expression and activity. For a pilot experiment, we suggest a concentration range from 20 µg to 80 µg of total protein extract.
      3. For recombinant protein study, active His-tagged OST1 protein kinase produced in E. coli was used (autophosphorylated OST1/SnRK2.6-6HIS, Vlad et al., 2009; see Notes section for recombinant protein purification). Different recombinant OST1/SnRK2.6-6HIS amounts where tested: 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 µg.
    9. Incubate loading samples on ice for 15 min.
    10. Load and run SDS-PAGE over-night (10-12 h) at constant 40 V.
      Notes:
      1. Optionally run SDS-PAGE for 4-5 h at constant 80 V.
      2. Run a parallel SDS-PAGE (without ∆C-ABF2 peptide) and load the same sample amount. After running, stain this gel with Coomassie to check protein loaded into gel. This Coomassie stained gel is also useful for densitometric analysis if required.

  3. In-gel kinase activity reaction
    Note: This protocol utilizes radioactive material and operators require experience in personal and environmental radiation protection and in the management of radioactive waste. (Important)
    1. Gel washing: wash gel 3 times (30 min each) with 100 ml of washing buffer (Recipe 5) at room temperature and gentle agitation.
    2. Protein renaturalization: incubate gel 3 times (first 2 times 30 min each, and the last incubation over-night 10-12 h) with 150 ml of protein renaturalization buffer (Recipe 6) at room temperature. Gentle agitation is required.
    3. Pre-reaction incubation: incubate gel 30 min in 40 ml of cold reaction kinase buffer (Recipe 7), at room temperature and with gentle agitation.
    4. Kinase reaction activity: incubate gel 1 h in 8 ml of hot reaction kinase buffer (Recipe 8). Gentle agitation and room temperature are required.
      Note: Prepare the minimum quantity possible of hot reaction kinase buffer to reduce radioactive waste.
    5. Kinase STOP reaction: wash gel 6 times (1 h each) with STOP-washing buffer (Recipe 9). Gentle agitation and room temperature are required.
    6. Gel drying: dry gel over a filter paper with a gel vacuum dryer.
    7. Radioactivity detection: detect radioactivity using screen and cassette (FUJIFILM) with variable exposure times (from 1 to 72 h) and a phosphorimage detector system (FLA5100; FUJIFILM).

Data analysis

In Figure 1 we show activity of His-tagged OST1 protein kinase (OST1/SnRK2.6-6HIS; Vlad et al., 2009) produced in E. coli. This kinase became autophosphorylated and active in absence of PP2Cs phosphatases. As expected, we see an increment in OST1 activity when we load more amount of the purified protein sample. On the other hand, we monitored the in vivo activation status of SnRK2s using crude protein extracts from wild-type (Col-0) and mutant lines: pyr/pyl112458 and snrk2.2/2.3/2.6 (Figure 2). The ‘In Gel Kinase Assay’ show kinase activity in the three ABA-activated SnRK2s (SnRK2.2/2.3/2.6), identified as a double band between 42 to 44 kD, in ABA-treated (100 µM ABA) Col-0. By contrast, as expected, this SnRK2s kinase activity was absent in control conditions Col-0 (mock treated) and in the ABA-treated (100 µM ABA) mutant line snrk2.2/2.3/2.6. Interestingly, the ‘In Gel Kinase Assay’ did not detect any activation of the SnRK2s in the sextuple mutant line pyr/pyl112458 after 100 µM ABA treatment. This result supports the idea that PYR/PYL112458 receptors are the main players in ABA perception and signaling.
 

Figure 1. Kinase activity measurement of recombinant OST1/SnRK2.6-6HIS. OST1/SnRK2.6-6HIS was produced in E. coli and purified by Ni affinity. Different recombinant protein amount where tested: 0.01, 0.05, 0.1, 0.5, 1, 5 and 10 µg. 1 h and 72 h: exposure time.


Figure 2. In vivo monitoring of SnRK2s activity. Wild-type (Col-0) and mutant lines: pyr/pyl112458 and snrk2.2/2.3/2.6 were mock or ABA (100 µM) treated for 30 min. A. SnRK2.2/2.3/2.6 kinase activity is detectable in ABA-treated (100 µM ABA) Col-0 and it is not detected in Col-0 control conditions (mock treated) neither ABA-treated (100 µM ABA) mutant line snrk2.2/2.3/2.6 nor sextuple mutant line pyr/pyl112458. Recombinant OST1/SnRK2.6-6HIS was used as a positive control. B. A parallel Coomassie stained SDS-PAGE. MW: molecular weight [kD]: 72, 55 and 43. RuBP-L: RuBisCO large chain.

Note: Given the necessity, substrate phosphorylation by SnRK2.2/2.3/2.6 over time can be quantified by densitometric analysis using open source software (e.g., ImageJ). Band signal volume (band area x band total intensity) in the phosphorylation assay gel must be calculated and the protein loading in the SDS-PAGE should be checked (a parallel Coomassie stained SDS-PAGE is an option).

Notes

  1. This technique does not present major problems in its execution, beyond those related to the radioactive ATP handling. To make a parallelism, the success in this technique may be similar to that obtained in Western-blots. In particular, the success rate of ‘In Gel Kinase Assay’ measuring SnRK2.2/2.3/2.6 kinase activity may be similar to the Western-blot technique using a good primary antibody.
  2. The triple mutant snrk2.2/2.3/2.6 was used as a negative control (no ABA-induced SnRK2s kinase activity). For snrk2.2, snrk2.3, snrk2.6 single mutant, snrk2.2/2.3 double mutant and snrk2.2/2.3/2.6 triple mutant characterization read Fujii et al. (2007 and 2009).
  3. Brief protocol for small-scale recombinant protein purification (from Antoni et al., 2012):
    1. E. coli BL21 (DE3) cells transformed with the corresponding constructs (OST1/SnRK2.6-6HIS and ∆C-ABF2, both in pET28a E. coli expression plasmid) were grown in 100 ml of LB medium to an optical density at 600 nm of 0.6 to 0.8.
    2. At this point, 1 mM isopropyl-b-D-thiogalactoside (IPTG) was added, and the cells were harvested after overnight incubation at 20 °C.
    3. Pellets were resuspended in lysis buffer (50 mM Tris, pH 7.5, 250 mM KCl, 10% [v/v] glycerol, and 1 mM b-mercaptoethanol) and lysed by sonication with a Branson Sonifier 250.
    4. The clear lysate obtained after centrifugation was purified by Ni affinity.
    5. A washing step was performed using 50 mM Tris, 250 mM KCl, 20% (v/v) glycerol, 30 mM imidazole, and 1 mM b-mercaptoethanol washing buffer, and finally the protein was eluted using 50 mM Tris, 250 mM KCl, 20% (v/v) glycerol, 250 mM imidazole, and 1 mM b-mercaptoethanol elution buffer.
      For an extended protocol of ‘Recombinant Protein Expression and Purification’ see this Bio-Rad protocol: http://www.bio-rad.com/webroot/web/pdf/lse/literature/pepsi_hr_1665067.pdf

Recipes

  1. Seed sterilization solution
    70% (v/v) ethanol
    0.05% (v/v) Tween-20
  2. Solid MS
    4.33 g/L Murashige & Skoog basal salts
    0.1% (w/v) MES
    1% (w/v) sucrose
    1% (w/v) agar
    Adjust pH to 5.7 with KOH before autoclaving
  3. Stock reagent
    1. 1 M Tris-HCl (pH 6.8 and 7.5), for 50 ml: 6.05 g Tris-base + 45 ml ddH2O. Bring to pH 6.8 or 7.5 with HCl and adjust volume with ddH2O to 50 ml (store at room temperature)
    2. 1 M HEPES-KOH (pH 7.5), for 50 ml: 14.2 g HEPES + 45 ml ddH2O. Bring to pH 7.5 with KOH and adjust volume with ddH2O to 50 ml (store at room temperature)
    3. 1 M DTT, for 50 ml: 7.5 g DTT in 50 ml ddH2O (store in aliquots at -20 °C)
    4. 0.5 M EGTA, for 50 ml: 9.5 g EGTA + 45 ml ddH2O. Bring to pH 8 with NaOH and adjust volume with ddH2O to 50 ml (store at room temperature)
    5. 0.5 M EDTA, for 50 ml: 9.3 g EDTA + 45 ml ddH2O. Bring to pH 8 with NaOH and adjust volume with ddH2O to 50 ml (store at room temperature)
    6. 1 M NaF, for 50 ml: 2.1 g NaF and adjust volume with ddH2O to 50 ml (store at 4 °C)
    7. 100 mM Na3VO4, for 100 ml: 1.82 g Na3VO4 + 90 ml ddH2O. Bring to pH 9 with HCl and boil solution until colorless. Cold solution to room temperature, adjust again to pH 9 with HCl and boil solution again (repeat this process until pH remain stable at pH 9 at room temperature). Adjust volume with ddH2O to 50 ml (store at -20 °C)
    8. 100 mM PMSF, for 10 ml: 174 mg PMSF adjust volume with ethanol to 10 ml (store in aliquots at -20 °C)
    9. 1 M MgCl2, for 10 ml: 4.76 g MgCl2 adjust volume with ddH2O to 10 ml (store in aliquots at -20 °C)
    10. 100 mM ATP, for 10 ml: 551 mg ATP adjust volume with ddH2O to 10 ml (store in aliquots at -20 °C)
  4. Protein extraction buffer
    5 mM EDTA
    5 mM EGTA
    2 mM DTT
    25 mM NaF
    1 mM Na3VO4
    50 mM BGP
    20% (v/v) glycerol
    1 mM PMSF
    1 tablet/100 ml protease inhibitor cocktail
    50 mM HEPES-KOH (pH 7.5)
  5. 2x Laemli buffer
    125 mM Tris-HCl, pH 6.8
    4% (w/v) SDS
    20% (v/v) glycerol
    0.001% bromo-phenol-blue
  6. Washing buffer
    25 mM Tris-HCl (pH 7.5)
    0.1 % (v/v) Triton X-100
    0.05 % (w/v) BSA
    0.5 mM DTT
    0.1 mM Na3VO4
    5 mM NaF
  7. Protein renaturalization buffer
    25 mM Tris-HCl (pH 7.5)
    1 mM DTT
    0.1 mM Na3VO4
    5 m M NaF
  8. Cold reaction kinase buffer
    40 mM HEPES-KOH (pH 7.5)
    1 mM EGTA
    200 mM MgCl2
    2 mM DTT
    0.1 mM Na3VO4
  9. Hot reaction kinase buffer
    8 ml cold reaction kinase buffer
    25 µM cold ATP (no radioactive)
    80 µCi hot ATP (radioactive)
  10. STOP-washing buffer
    5% (w/v) TCA (trichloroacetic acid)
    1% (p/v) Na2PPi (sodium pyrophosphate)

Acknowledgments

We thank Prof. Christiane Laurière for giving us kind assistance in the development of this protocol. This work was supported by the Ministerio de Ciencia e Innovación, Fondo Europeo de Desarrollo Regional, and Consejo Superior de Investigaciones Científicas (grant BIO2014-52537-R to P.L.R). This protocol was adapted from the research article of Gonzalez-Guzman et al. (2012). The author declares no conflicts of interest.

References

  1. Antoni, R., Gonzalez-Guzman, M., Rodriguez, L., Rodrigues, A., Pizzio, G. A. and Rodriguez, P. L. (2012). Selective inhibition of clade A phosphatases type 2C by PYR/PYL/RCAR abscisic acid receptors. Plant Physiol 158(2): 970-980.
  2. Antoni, R., Rodriguez, L., Gonzalez-Guzman, M., Pizzio, G. A. and Rodriguez, P. L. (2011). News on ABA transport, protein degradation, and ABFs/WRKYs in ABA signaling. Curr Opin Plant Biol 14(5): 547-553.
  3. Dupeux, F., Antoni, R., Betz, K., Santiago, J., Gonzalez-Guzman, M., Rodriguez, L., Rubio, S., Park, S. Y., Cutler, S. R., Rodriguez, P. L. and Marquez, J. A. (2011). Modulation of abscisic acid signaling in vivo by an engineered receptor-insensitive protein phosphatase type 2C allele. Plant Physiol 156(1): 106-116.
  4. Fujii, H., Verslues, P. E. and Zhu, J. K. (2007). Identification of two protein kinases required for abscisic acid regulation of seed germination, root growth, and gene expression in Arabidopsis. Plant Cell 19(2): 485-494.
  5. Fujii, H. and Zhu, J. K. (2009). Arabidopsis mutant deficient in 3 abscisic acid-activated protein kinases reveals critical roles in growth, reproduction, and stress. Proc Natl Acad Sci U S A 106(20): 8380-8385.
  6. 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.
  7. He, F. (2011). Laemmli-SDS-PAGE. Bio-protocol Bio101: e80.
  8. Vlad, F., Rubio, S., Rodrigues, A., Sirichandra, C., Belin, C., Robert, N., Leung, J., Rodriguez, P. L., Lauriere, C. and Merlot, S. (2009). Protein phosphatases 2C regulate the activation of the Snf1-related kinase OST1 by abscisic acid in Arabidopsis. Plant Cell 21(10): 3170-3184.

简介

正常发育和体内平衡需要细胞和组织中蛋白质磷酸化的适时时空调节。我们提出方案“凝胶激酶测定”,其可用于粗蛋白质提取物的蛋白激酶活性测量。我们已经成功地使用“凝胶激酶测定”方案来证明在ABA受体(PYR / PYL / RCAR-ABA受体)的PYRABACTIN RESISTANCE1 / PYR1-样/调节组分中的拟南芥线条pyr / pyl112458 )在ABA介导的SnRK2.2,SnRK2.3和OST1 / SnRK2.6的活化中受损,多达三重突变体snrk2.2 / 2.3 / 2.6 (Gonzalez-Guzman等人,2012)。

背景 植物激素脱落酸(ABA)是涉及植物生长发育以及植物对非生物和生物胁迫的反应的关键信号。 ABA感知和信号通路由ABA受体(PYR / PYL / RCAR-ABA受体)的PYRABACTIN RESISTANCE1 / PYR1-调节组分,PP2C磷酸酶和SnRK2s激酶组成(在Antoni等人, ,,2011)。模块受体-ABA-磷酸酶通过调节ABA激活的SnRK2而以配体依赖的方式控制磷酸化信号级联。反过来,SnRK2s激酶使细胞核和细胞质中的无数效应物从转录因子(例如,ABFs)到离子通道(例如)磷酸化, SLAC1)。我们在这里详细介绍了“凝胶激酶测定”方案。该方案开发用于植物组织蛋白提取物以及纯化的重组激酶中的蛋白激酶活性测定。简言之:制备含有ΔC-ABF2肽(特异性SnRK2.2,SnRK2.3,OST1 / SnRK2.6激酶底物)的SDS-聚丙烯酰胺凝胶。 ΔC-ABF2肽被捕获在SDS-聚丙烯酰胺凝胶网中,并且在电泳过程中不迁移。另一方面,不经煮沸而没有任何还原剂(例如,DTT或B-ME)的经SDS处理的蛋白质样品仅部分变性并且可以在一定程度上被再归化洗出SDS。此外,可以使用[Gamma-32p] ATP测量高敏感水平的激酶活性。以这种方式,与激酶活性值一起,我们也具有与激酶活性相关的电泳迁移率值。使用植物粗蛋白提取物,在样品电泳和凝胶蛋白变性步骤后,我们可以测定凝胶中特定的SnRK2.2 / 2.3 / 2.6(SnRK2s)激酶活性。我们已经应用该方案来表征PYR / PYL / RCAR-ABA受体六联体突变体中的SnRK2s活性。我们显示,在ABA介导的SnRK2的激活中,六重突变体突变体系pyr / pyl112458 受损,与三突变体snrk2.2 / 2.3 / 2.6中的三重突变体>拟南芥(结果发表在冈萨雷斯 - 古斯曼等人,2012)。

关键字:激酶, 磷酸化, 体内, 酶活性, SnRKs, 脱落酸, 拟南芥

材料和试剂

  1. Eppendorf和Falcon管(分别为1.5ml和50ml;通用)
  2. 移液器和提示(通用)
  3. Micropore磁带(3M,目录号:1530-0)
  4. (100毫米直径x 20毫米高度;通用的:例如,Greiner Bio One,目录号:664161)
  5. 7.5 x 10 cm的超厚印迹滤纸(Bio-Rad Laboratories,目录号:1703965)
  6. 拟南芥种子:Col-0(NASC,N1093),pyr/pyl112458和/或snrk2.2/2.3/2.6(冈萨雷斯 - 古斯曼(Gonzalez-Guzman) em> et al。,2012)
  7. 可选:纯化的重组激酶OST1(SnRK2.6-6his;自制:Vlad et al。,2009和2012)
  8. 无菌去离子水(通用)
  9. 液体N 2(通用)
  10. 冰(通用)
  11. ABA(+顺式,反式脱落酸)(BIOSYNTH,目录号:A-0120)
  12. Bio-Rad蛋白测定试剂盒I(Bio-Rad Laboratories,目录号:5000001)
  13. Bio-Safe TM考马斯染色试剂(Bio-Rad Laboratories,目录号:1610786)
  14. ΔC-ABF2肽(氨基酸1至173; 18.4KD;自制:Dupeux等人,2011; Antoni等人,2012)
    注意:市售替代MBP(髓磷脂碱性蛋白)(Sigma-Aldrich,目录号:M1891)。
  15. SDS-PAGE试剂(列于He,2011)
  16. 乙醇(通用)
  17. 聚乙二醇脱水山梨醇单月桂酸酯(吐温-20)(Sigma-Aldrich,目录号:P1379)
  18. Murashige和Skoog培养基(MS)(PhytoTechnology Laboratories ®,目录号:M524)
  19. 2-N-吗啉代 - 乙磺酸(MES)(Sigma-Aldrich,目录号:M8250)
  20. 蔗糖(VWR,BDH ®,目录号:BDH0308)
  21. 琼脂(Sigma-Aldrich,目录号:A1296)
  22. 氢氧化钾(KOH)(Fisher Scientific,目录号:P250)
  23. 2-氨基-2-(羟甲基)-1,3-丙二醇(Tris-碱)(Sigma-Aldrich,目录号:252859)
  24. HCl
  25. 4-(2-羟乙基)哌嗪-1-乙磺酸,N-(2-羟乙基)哌嗪-N' - (2-乙磺酸)(HEPES)(Sigma-Aldrich,目录号:H3375)
  26. DL-二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)
  27. EDTA(Sigma-Aldrich,目录号:E5134)
  28. EGTA(Sigma-Aldrich,目录号:E3889)
  29. 氟化钠(NaF)(Sigma-Aldrich,目录号:S7920)
  30. 原钒酸钠(Na 3 VO 4)(Sigma-Aldrich,目录号:450243)
  31. 苯甲基磺酰氟(PMSF)(Sigma-Aldrich,目录号:P7626)
  32. 氯化镁(MgCl 2)(Sigma-Aldrich,目录号:M8266)
  33. 腺苷5'-三磷酸二钠水合物(ATP)(Sigma-Aldrich,目录号:A1852)
  34. 甘油2-磷酸二钠盐水合物(BGP)(Sigma-Aldrich,目录号:G9422)
  35. 甘油(Sigma-Aldrich,目录号:G5516)
  36. 蛋白酶抑制剂混合物(Sigma-Aldrich,目录号:S8830)
  37. 十二烷基硫酸钠(SDS)(Sigma-Aldrich,目录号:L3771)
  38. 2-(5-溴-2-吡啶基偶氮)-5-(二乙基氨基)(溴 - 苯酚蓝)(Sigma-Aldrich,目录号:180017)
  39. 聚乙二醇叔辛基苯基醚(Triton X-100)(Sigma-Aldrich,目录号:T8787)
  40. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:05470)
  41. 10μg/ml-3,000Ci/mmol(PerkinElmer,目录号:BLU502A)
  42. 三氯乙酸(TCA)(Sigma-Aldrich,目录号:T6399)
  43. 焦磷酸钠(Na 2 PPi)(Sigma-Aldrich,目录号:71501)
  44. 种子灭菌方案(见食谱)
  45. 固体MS(参见食谱)
  46. 库存试剂(参见食谱)
    1. 1M Tris-HCl(pH 6.8和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. 100mM Na 3 VO 4< 4>
    8. 100 mM PMSF
    9. 1 M MgCl 2
    10. 100 mM ATP
  47. 蛋白质提取缓冲液(参见食谱)
  48. 2x Laemli缓冲液(见配方)
  49. 洗涤缓冲液(见配方)
  50. 蛋白质变性缓冲液(参见食谱)
  51. 冷反应激酶缓冲液(见配方)
  52. 热反应激酶缓冲液(见配方)
  53. 停止洗涤缓冲液(见配方)

设备

  1. 辐射安全装备和个人防护(通用)
  2. 移液器
  3. 流通罩(通用型:例如,,CLEATECH,目录号:1000-11-E)
  4. 植物生长室(通用:例如,BioChambers,型号:FXC-9)
  5. 台式微量离心机(通用型:例如,Thermo Fisher Scientific,Thermo Scientific TM ,型号:Sorvall TM Legend TM Micro 17,目录号:75002430)
  6. SDS-PAGE系统(Bio-Rad Laboratories,型号:Mini-PROTEAN Tetra Vertical Electrophoresis Cell)
  7. 真空干燥器(通用型:例如Bio-Rad Laboratories,型号:583 Gel Dryers)
  8. 磷光显微镜屏幕和磁带(FUJIFILM)
  9. 磷光计(FUJIFILM,型号:FLA-5100)
  10. 高压灭菌(通用)
  11. 砂浆(100毫米直径x 60毫米高度;通用)
  12. 痉挛和镊子(通用)
  13. 塑料孵化箱(用于聚丙烯酰胺凝胶孵育和洗涤;通用)
  14. 搅拌棒(50毫米长;通用)
  15. 计时器(通用)
  16. 旋转振动筛(通用)
  17. 冰箱(4°C;通用型)
  18. 冰柜(-20°C和-80°C;通用型)
  19. 磁力搅拌器(通用)
  20. pH计(通用:例如,Thermo Fisher Scientific,Thermo Scientific TM,型号:Orion Star A111)
  21. 余额(通用型:例如,,Sartorius,型号:Cubis ®精准天平)
  22. 漩涡(通用)
  23. 分光光度计(通用:例如Thermo Fisher Scientific,Thermo Scientific TM,型号:Evolution TM 300)

软件

  1. ImageJ(可选)

程序

注意:尽管该方案是针对植物组织蛋白提取物以及重组蛋白而开发的,但也可用于任何其他来源的蛋白质提取物。更重要的是,该方案对于从免疫沉淀测定获得的样品也是有效的(Vlad。,et al。,2009)。

  1. 种子灭菌和植物生长
    1. 将5mg(200-300粒种子)的拟南芥种子放入Eppendorf管中,加入750μl灭菌溶液(方案1),并在室温下摇动20分钟。
      注意:在本协议中,我们测试:pyr/pyl sextuple突变体系(pyr/pyl112458),snrk三重突变体系(snrk2.2/2.3/2.6)和Col-0作为对照品系(Gonzalez-Guzman et al。,2012)。
    2. 在流动罩中取出灭菌溶液(用无菌尖端吸移),用750μl无菌蒸馏水洗涤种子(室温下振荡15分钟)。重复此步骤3次。
    3. 在750微升新鲜的无菌蒸馏水中,在4℃下将种子分层在黑暗中3天
    4. 在无菌固体MS板上播种种子(配方2)。
      注意:保持每厘米大约一个种子的密度(拥挤的板块产生不健康的幼苗)。
    5. 用微孔胶带封闭并密封。
      注意:只能使用一层微孔磁带。不止一层防止气体交换和健康植物生长。
    6. 在受控环境生长室中,在22℃,16小时光照/8小时暗光照条件下,以100μEm-2 /秒的速度孵育平板12天。
      注意:时间和成长条件可能根据实验要求而变化。

  2. 总蛋白提取和SDS-PAGE
    1. 从板上收获幼苗(500mg)并将其冷冻在液体N 2中。储存于-80°C直至使用。
      注意:在我们的实验设计中,在冷冻之前需要100μAABA或模拟处理(喷雾-30分钟)。
    2. 使用砂浆和液体N 2将植物组织研磨成细粉。
    3. 使用冷却(N 2)刮刀将400mg细粉末转移到2ml Eppendorf管中;加入1ml冷提取缓冲液(配方3),并通过涡旋15秒混合。
      注意:将管保持在冰中,直到离心(继续下一个样品)。
    4. 在4℃离心15分钟(最高速度,14,000 x g )。
    5. 将上清液转移到新的1.5 ml Eppendorf管中,储存于-80°C直至使用。
      注意:在-80°C储存管之前,取每个样品(50μl)进行蛋白质定量。重复样品冻结和解冻可以降低酶活性。
    6. 继续进行蛋白质定量。
      注意:任何蛋白质定量方法都应该是好的。我们选择Bio-Rad的Bradford蛋白质测定法(遵循制造商协议)
    7. 准备10%聚丙烯酰胺凝胶(用于SDS-PAGE),并在聚合前包括800μg的ΔC-ABF2肽(在分辨凝胶中)。
      注意:
      1. 我们选择Bio-Rad的miniprotean III SDS-PAGE系统和1.5 mm宽的凝胶。
      2. 我们使用特异性SnRK2.2/2.3/.2.6激酶底物His6-ΔC-ABF2肽(氨基酸1至173; 18.4KD)。 ΔC-ABF2克隆和重组蛋白质制备描述于:Dupeux等人,2011和Antoni等人,2012(参见Notes部分中这些方法的简要说明)。
      3. 可以使用潜在的其它重组激酶底物,取决于将测定哪些激酶。甚至合成或商业上可获得的肽,即MBP(髓磷脂碱性蛋白)也应该是合适的。
      4. 对于SDS-PAGE制备,遵循BIOPROTOCOL e80(He,2011)
    8. 准备加载样品:用50μl2x Laemli缓冲液(方案5)将40μg总蛋白质植物提取物(在步骤B5中获得并在冰上解冻)混合,并加入无菌去离子H 2 O以达到100μl最终卷。
      注意:
      1. 不要煮沸样品。沸腾产生不可逆的蛋白质变性和酶活性的丧失。 (重要)
      2. 总蛋白提取物的量可以根据激酶表达和活性而变化。对于试点实验,我们建议浓度范围为20μg至80μg总蛋白提取物。
      3. 对于重组蛋白质研究,使用在大肠杆菌中产生的活性His标记的OST1蛋白激酶(自磷酸化OST1/SnRK2.6-6HIS,Vlad等人,2009;参见关于重组蛋白纯化的注释部分)。测试的不同重组OST1/SnRK2.6-6HIS量:0.01,0.05,0.1,0.5,1,5和10μg。
    9. 孵育加载样品在冰中15分钟
    10. 加载并运行SDS-PAGE(10-12小时),恒定40V。
      注意:

      1. 可选择在80 V恒定运行SDS-PAGE 4-5 h
      2. 运行平行SDS-PAGE(不含ΔC-ABF2肽)并加载相同的样品量。运行后,用考马斯染色该凝胶以检查加载到凝胶中的蛋白质。如果需要,考马斯染色的凝胶也可用于光密度分析

  3. 凝胶内激酶活性反应
    注意:本协议使用放射性物质,操作人员需要个人和环境辐射防护经验和放射性废物管理经验。 (重要)
    1. 凝胶洗涤:在室温和温和搅拌下,用100ml洗涤缓冲液(配方5)洗涤凝胶3次(每次30分钟)。
    2. 蛋白质变性:在室温下,用150ml蛋白质变性缓冲液(食谱6)孵育凝胶3次(每次2次,每次10分钟,最后一次孵育10-12小时)。需要温和的激动。
    3. 预反应孵育:在室温和温和搅拌下,在40ml冷反应激酶缓冲液(食谱7)中孵育30分钟。
    4. 激酶反应活性:在8 ml热反应激酶缓冲液(配方8)中孵育凝胶1 h。需要温和的搅拌和室温。
      注意:准备尽可能多的热反应激酶缓冲液以减少放射性废物。
    5. 激酶停止反应:用停止洗涤缓冲液(食谱9)洗涤凝胶6次(每次1小时)。需要温和的搅拌和室温。
    6. 凝胶干燥:用凝胶真空干燥器在滤纸上干燥凝胶
    7. 放射性检测:使用具有可变曝光时间(1至72小时)和荧光图像检测器系统(FLA5100; FUJIFILM)的屏幕和盒(FUJIFILM)来检测放射性。

数据分析

在图1中,我们显示了E标记的His标记的OST1蛋白激酶(OST1/SnRK2.6-6HIS; Vlad。等人,2009)的活性。大肠杆菌。这种激酶在没有PP2Cs磷酸酶的情况下自发磷酸化并且是活性的。如预期的那样,当我们加载更多量的纯化蛋白质样品时,我们看到OST1活性增加。另一方面,我们使用来自野生型(Col-0)和突变体系的粗蛋白提取物监测SnRK2s的体内活化状态:pyr/pyl112458和< em> snrk2.2/2.3/2.6 (图2)。在ABA处理的(100μMABA)Col-0中,"In Gel Kinase Assay"显示在被鉴定为42至44kD之间的双重带的三种ABA活化的SnRK2(SnRK2.2/2.3/2.6)中的激酶活性。相反,如预期的那样,在对照条件Col-0(模拟处理)和ABA处理的(100μMABA)突变体株系snrk2.2/2.3/2.6中,SnRK2s激酶活性不存在。有趣的是,在100μMABA处理后,"In Gel Kinase Assay"没有检测出在六色子突变体系pyr/pyl112458中的SnRK2的任何活化。该结果支持PYR/PYL112458受体是ABA感知和信号传导的主要参与者。
 

图1.重组OST1/SnRK2.6-6HIS的激酶活性测定。 OST1/SnRK2.6-6HIS在E中产生。大肠杆菌,并通过Ni亲和力纯化。测试的不同重组蛋白质量:0.01,0.05,0.1,0.5,1,5和10μg。 1 h和72 h:曝光时间。


图2. 监测SnRK2s活性野生型(Col-0)和突变体系:pyr/pyl112458 和 snrk2.2/2.3/2.6 被模拟或ABA(100μM)处理30分钟。 A.ArRK2.2/2.3/2.6激酶活性在ABA处理(100μMABA)Col-0中是可检测的,并且在Col-0对照条件(模拟处理)中没有检测到ABA处理(100μMABA)突变体线 snrk2.2/2.3/2.6 也不是sextuple突变体线pyr/pyl112458 。重组OST1/SnRK2.6-6HIS用作阳性对照。 B.平行的Coomasie染色SDS-PAGE。 MW:分子量[kD]:72,55和43. RuBP-L:RuBisCO大链
注意:鉴于必要性,SnRK2.2/2.3/2.6随时间的底物磷酸化可以通过使用开源软件(例如ImageJ)的光密度分析进行定量。必须计算磷酸化测定凝胶中的带信号体积(带区x带总强度),并检查SDS-PAGE中的蛋白质负载量(并行考马斯染色的SDS-PAGE是一种选择)。

笔记

  1. 这种技术在执行中不存在重大问题,超出与放射性ATP处理有关的问题。为了平行化,这种技术的成功可能类似于在西方国家获得的技术。特别地,测定SnRK2.2/2.3/2.6激酶活性的"凝胶激酶测定"的成功率可能与使用良好的一级抗体的Western印迹技术相似。
  2. 三重突变体snrk2.2/2.3/2。 6 被用作阴性对照(没有ABA诱导的SnRK2s激酶活性)。对于 snrk2.2 snrk2.3 , snrk2.6 单突变体, snrk2.2/2.3 双突变体和 snrk2.2/2.3/2.6 三重突变体特征读取Fujii等人(2007年和2009年)。
  3. 小规模重组蛋白纯化的简要方案(来自Antoni等人,2012):
    1. E。用对应的构建体(OST1/SnRK2.6-6HIS和ΔC-ABF2,都在pET28a大肠杆菌表达质粒中)转化的大肠杆菌BL21(DE3)细胞在100毫升的LB培养基至600nm的光密度为0.6〜0.8。
    2. 此时,加入1mM异丙基-b-D-硫代半乳糖苷(IPTG),并在20℃孵育过夜后收获细胞。
    3. 将颗粒重悬于裂解缓冲液(50mM Tris,pH 7.5,250mM KCl,10%[v/v]甘油和1mM b-巯基乙醇)中,并用Branson Sonifier 250超声处理裂解。
    4. 离心后得到的澄清裂解物用Ni亲和力纯化
    5. 使用50mM Tris,250mM KCl,20%(v/v)甘油,30mM咪唑和1mMβ-巯基乙醇洗涤缓冲液进行洗涤步骤,最后使用50mM Tris,250mM KCl ,20%(v/v)甘油,250mM咪唑和1mM b-巯基乙醇洗脱缓冲液。
      对于"重组蛋白表达和纯化"的扩展方案,请参阅Bio-Rad协议: http://www.bio-rad.com/webroot/web/pdf/lse/literature/pepsi_hr_1665067.pdf。

食谱

  1. 种子灭菌解决方案
    70%(v/v)乙醇 0.05%(v/v)Tween-20
  2. 固体MS
    4.33 g/L Murashige& Skoog基础盐
    0.1%(w/v)MES
    1%(w/v)蔗糖 1%(w/v)琼脂
    在高压灭菌前用KOH调节pH至5.7
  3. 库存试剂
    1. 1M Tris-HCl(pH6.8和7.5),50ml:6.05g Tris-碱+ 45ml ddH 2 O。用HCl调至pH 6.8或7.5,并用ddH 2 O调节体积至50ml(室温下储存)
    2. 1M HEPES-KOH(pH7.5),50ml:14.2g HEPES + 45ml ddH 2 O。用KOH调至pH7.5并用ddH 2 O调节体积至50ml(室温下储存)
    3. 1 M DTT,50ml:7.5 g DTT,于50ml ddH 2 O(-20℃保存)等等。
    4. 0.5M EGTA,50ml:9.5g EGTA + 45ml ddH 2 O。用NaOH调至pH8,并用ddH 2 O调节体积至50ml(室温下储存)
    5. 0.5M EDTA,50ml:9.3g EDTA + 45ml ddH 2 O。用NaOH调至pH8,并用ddH 2 O调节体积至50ml(室温下储存)
    6. 1M NaF,50ml:2.1g NaF,并用ddH 2 O至50ml调节体积(在4℃下储存)
    7. 100毫摩尔Na 3 VO 4,100ml,1.82克Na 3 VO 4+ + 90毫升ddH, sub> 2 O。用HCl调至pH9,煮沸溶液至无色。冷至室温,用HCl再次调节至pH9,再次沸腾溶液(重复此过程直到pH在室温下保持稳定在pH9)。调整体积与ddH <2> O至50ml(-20℃保存)
    8. 100毫摩尔PMSF,10ml:174毫克PMSF调节体积用乙醇至10毫升(储存在等分-20℃)
    9. 1 M MgCl 2,10ml:4.76g MgCl 2调节体积,ddH 2 O至10ml(以等分试样储存于-20℃ °C)
    10. 100毫升ATP,10ml:551毫克ATP调节体积,ddH 2 O至10毫升(以等分试样-20℃保存)
  4. 蛋白质提取缓冲液
    5 mM EDTA
    5 mM EGTA
    2 mM DTT
    25 mM NaF
    1mM Na 3 VO 4< 4>
    50 mM BGP
    20%(v/v)甘油 1 mM PMSF
    1片/100毫升蛋白酶抑制剂鸡尾酒
    50mM HEPES-KOH(pH7.5)
  5. 2x Laemli缓冲区
    125mM Tris-HCl,pH6.8
    4%(w/v)SDS
    20%(v/v)甘油 0.001%溴酚蓝
  6. 洗涤缓冲液
    25mM Tris-HCl(pH7.5)
    0.1%(v/v)Triton X-100
    0.05%(w/v)BSA
    0.5 mM DTT
    0.1mM Na 3 VO 4< 4>
    5 mM NaF
  7. 蛋白质变性缓冲液
    25mM Tris-HCl(pH7.5)
    1 mM DTT
    0.1mM Na 3 VO 4< 4>
    5 m M NaF
  8. 冷反应激酶缓冲液
    40 mM HEPES-KOH(pH 7.5)
    1 mM EGTA
    200mM MgCl 2
    2 mM DTT
    0.1mM Na 3 VO 4< 4>
  9. 热反应激酶缓冲液
    8 ml冷反应激酶缓冲液
    25μM冷ATP(无放射性)
    80μCi热ATP(放射性)
  10. 停止清洗缓冲区
    5%(w/v)TCA(三氯乙酸)
    1%(p/v)Na 2 PPi(焦磷酸钠)

致谢

我们感谢ChristianeLaurière教授在本协议的制定中给予我们善意的协助。这项工作得到Ministry of de Ciencia eInnovación,Fondo Europeo de Desarrollo Regional和Consejo Superior de InvestigacionesCientíficas(授予BIO2014-52537-R至P.L.R)的支持。该协议是由冈萨雷斯 - 古斯曼等人的研究文章改编而成。 (2012)。作者声明没有利益冲突。

参考文献

  1. Antoni,R.,Gonzalez-Guzman,M.,Rodriguez,L.,Rodrigues,A.,Pizzio,GA和Rodriguez,PL(2012)。  通过PYR/PYL/RCAR脱落酸受体选择性抑制2C型磷酸酶。植物生理学 em> 158(2):970-980。
  2. Antoni,R.,Rodriguez,L.,Gonzalez-Guzman,M.,Pizzio,GA和Rodriguez,PL(2011)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/21742545"target ="_ blank"> ABA传输,蛋白质降解和ABA信号中的ABF/WRKY的新闻。Curr Opin Plant B iol 14(5):547-553。
  3. Dupeux,F.,Antoni,R.,Betz,K.,Santiago,J.,Gonzalez-Guzman,M.,Rodriguez,L.,Rubio,S.,Park,SY,Cutler,SR,Rodriguez,PL and Marquez ,JA(2011)。  脱落酸信号的调制通过工程化的受体不敏感蛋白磷酸酶2C型等位基因的体内实现。植物生理学156(1):106-116。
  4. Fujii,H.,Verslues,PE and Zhu,JK(2007)。  鉴定拟南芥中种子萌发,根生长和基因表达的脱落酸调节所需的两种蛋白激酶。植物细胞 19(2):485-494。 br />
  5. Fujii,H. and Zhu,JK(2009)。  在3种脱落酸激活的蛋白激酶中缺失突变体的拟南芥突变体在生长,繁殖和应激中显示出关键作用。美国Proc Natl Acad Sci USA 106(20): 8380-8385。
  6. Gonzalez-Guzman,M.,Pizzio,GA,Antoni,R.,Vera-Sirera,F.,Merilo,E.,Bassel,GW,Fernandez,MA,Holdsworth,MJ,Perez-Amador,MA,Kollist,和Rodriguez,PL(2012)。 拟南芥 PYR/PYL/RCAR受体在气孔孔径的定量调节和对脱落酸的转录反应中起主要作用。植物细胞24(6):2483-2496。 br />
  7. 他,F.(2011)。 Laemmli-SDS-PAGE。 Bio> Bio101 Bio101:e80。
  8. Vlad,F.,Rubio,S.,Rodrigues,A.,Sirichandra,C.,Belin,C.,Robert,N.,Leung,J.,Rodriguez,PL,Lauriere,C.and Merlot,S。(2009 )。蛋白磷酸酶2C调节Snf1-相关的激酶OST1通过拟南芥中的脱落酸进行。植物细胞 21(10):3170-3184。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
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