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In vitro NLK Kinase Assay

体外NLK激酶测定

SM Sungho Moon *
JK Jiyoung Kim *
Eek‐hoon Jho Eek‐hoon Jho

 (*contributed equally to this work) 发布: 2017年11月05日第7卷第21期 DOI: 10.21769/BioProtoc.2593 浏览次数: 11564

评审: Ralph BottcherAnonymous reviewer(s)

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Abstract

This protocol provides step by step instructions to perform an in vitro kinase assay for nemo-like kinase. In addition, this protocol also describes an efficient method using mild lysis buffer for expression and purification of Glutathione S-transferase (GST) fusion proteins.

Keywords: NLK (NLK) search in vitro kinase assay (体外激酶测定) search GST-fusion protein (GST融合蛋白) search YAP (YAP) search Hippo pathway (Hippo通路) search

Background

Drosophila nemo has been identified as a pivotal regulator of photoreceptor clusters in the developing Drosophila eyes (Choi and Benzer, 1994). It has been reported that nemo is conserved across various species. The mammalian homolog is nemo-like kinase (NLK) (Brott et al., 1998; Rocheleau et al., 1999). NLK is a member of the CMGC group (CDK, MAPK, GSK3 and CLK) and also belongs to the atypical MAPK family. NLK regulates multiple cellular mechanisms through transcription factors including TCF/LEF1, NICD, and FOXO, which are key players in diverse signaling pathways (Ishitani et al., 1999; Ishitani et al., 2010; Kim et al., 2010). Moreover, recently we have identified NLK as a novel kinase for Yes-associated protein (YAP), a co-transcriptional activator in the Hippo pathway (Moon et al., 2017). This protocol describes an in vitro method to measure the nemo-like kinase activity.

Materials and Reagents

  1. Pipette tips–1,000 μl tips (OHAUS, catalog number: PCB-1000 ), 200 μl tips (Neptune Scientific, catalog number: 2100 )
  2. 14 ml round bottom tubes (SPL, catalog number: 40014 )
  3. 15 ml conical centrifuge tubes (Ihanil Scientific, catalog number: LP-00015CP-00 )
  4. 60 mm cell culture dish (SPL, catalog number: 20060 )
  5. Microcentrifuge tube (SARSTEDT, catalog number: 72.690 )
  6. X-ray film cassette (Advansta, catalog number: L-07019-001 )
  7. X-ray film (XSANATEC, AGFA, catalog number: EA75Q )
  8. Cell scraper (SARSTEDT, catalog number: 83.1832 )
  9. Centrifuge bottles (Sigma-Aldrich, catalog number: B1033 )
  10. pGEX-4T-3 vector (GE Healthcare, catalog number: 28-9545-52 )
  11. pFLAG-CMV4 vector (Sigma-Aldrich, catalog number: E7158 )
  12. Flag-NLK vectors (Ishitani et al., 1999)
  13. Competent cells-BL21 (DE3) (EMD Millipore, Novagen, catalog number: 70235 )
  14. Homo sapiens (Human) HEK293 cells (ATCC, catalog number: CRL-1573 )
  15. Zinc chloride (ZnCl2) (Sigma-Aldrich, catalog number: 229997 )
  16. Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Bio Basic, catalog number: IB0168 )
  17. Glutathione Sepharose 4B (GE Healthcare, catalog number: 17075601 )
  18. Ethanol (Merk, catalog number: 1.00983.1011 )
  19. Bovine serum albumin (BSA) (Bio Basic, catalog number: AD0023 )
  20. Dulbecco’s modified Eagle medium (DMEM) (Lonza, catalog number: 12-604F )
  21. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 16000044 )
  22. Antibiotic-Antimycotic (Thermo Fisher Scientific, GibcoTM, catalog number: 15240062 )
  23. TurboFect transfection reagents (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: R0531 )
  24. Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )
  25. Anti-FLAG M2 antibody (Sigma-Aldrich, catalog number: F3165 )
  26. Protein A/G plus agarose (Santa Cruz Biotechnology, catalog number: sc-2003 )
  27. Adenosine 5’-triphosphate (ATP) [γ-32P] (PerkinElmer, catalog number: NEG502A )
  28. LB broth high salt (Duchefa Biochemie, catalog number: L1704 )
  29. Micro agar (Duchefa Biochemie, catalog number: M1002 )
  30. Ampicillin (Duchefa Biochemie, catalog number: A0104 )
  31. Tryptone (Duchefa Biochemie, catalog number: T1332 )
  32. Yeast extract (Duchefa Biochemie, catalog number: Y1333 )
  33. Sodium chloride (NaCl) (Duchefa Biochemie, catalog number: S0520 )
  34. Potassium chloride (KCl) (Duchefa Biochemie, catalog number: P0515 )
  35. Di-sodium hydrogen phosphate dihydrate (Na2HPO4·2H2O) (Duchefa Biochemie, catalog number: S0537 )
  36. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 105108 )
  37. Tris (Duchefa Biochemie, catalog number: T1501 )
  38. NP-40 (IGEPAL CA-630) (Sigma-Aldrich, catalog number: I8896 )
  39. Ethylenediaminetetraacetic acid (EDTA) (Duchefa Biochemie, catalog number: E0511 )
  40. Ethylene glycol-bis(2-aminoethylether)-N,N,N’,N’,-tetraacetic acid (EGTA) (MP Biomedicals, catalog number: 194823 )
  41. β-Glycerophosphate (MP Biomedicals, catalog number: 195206 )
  42. Sodium fluoride (NaF) (Sigma-Aldrich, catalog number: S1504 )
  43. Dithiothreitol (DTT) (Bio Basic, catalog number: DB0058 )
  44. Phenylmethylsulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  45. Leupeptin (Sigma-Aldrich, catalog number: L2884 )
  46. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
  47. L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
  48. Methanol (Merck, catalog number: 106099 )
  49. Acetic acid (Merck, catalog number: 100056 )
  50. Coomassie Blue (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20279 )
  51. HEPES (pH 7.5) (Duchefa Biochemie, catalog number: H1504.0100 )
  52. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  53. Sodium dodecyl sulfate (SDS) (Duchefa Biochemie, catalog number: S1377 )
  54. Glycerol (Duchefa Biochemie, catalog number: G1345.1000 )
  55. β-Mercaptoethanol (AMRESCO, catalog number: 0482 )
  56. Bromophenol Blue (BPB) (AMRESCO, catalog number: 0449 )
  57. Sodium orthovanadate (Sigma-Aldrich, catalog number: S6508 )
  58. Lysozyme (Agilent Technologies, catalog number: EC 3.2.1.17 )
  59. LB medium (see Recipes)
  60. LB agar plate (see Recipes)
  61. 2x YTA medium (see Recipes)
  62. 1x phosphate-buffered saline (PBS) (see Recipes)
  63. Cell lysis buffer A (see Recipes)
  64. Cell lysis buffer B (see Recipes)
  65. Elution buffer (see Recipes)
  66. Coomassie Blue staining buffer (see Recipes)
  67. 10x kinase buffer (see Recipes)
  68. 4x SDS loading dye (see Recipes)
  69. Destaining buffer (see Recipes)

Equipment

  1. Shaking incubator (VISION SCIENTIFIC, model: VS-8480SF )
  2. Erlenmeyer flask (Corning, PYREX®, catalog number: 4980-500 )
  3. UV spectrophotomter (Mecasys, Optizen, model: 2120UV )
  4. Centrifuge for 50/250 ml tubes
  5. 50 ml centrifuge tubes (Thermo Fisher Scientific, Thermo ScientificTM, model: 3119 )
  6. 250 ml centrifuge bottles (Thermo Fisher Scientific, Thermo ScientificTM, model: 3120 )
  7. Sonicator (Sonics & Materials, model: VCX 500 )
  8. High speed refrigerated micro centrifuge (TOMY DIGITAL BIOLOGY, model: MX-300 )
  9. Water-Jacketed CO2 incubator (Thermo Fisher Scientific, model: Forma Series II 3111 )
  10. Pipettes (Gilson, model: PIPETMAN®, catalog number: F167300 )
  11. Micro centrifuge rotor rack (TOMY DIGITAL BIOLOGY, model: AR015-24 )
  12. Micro centrifuge rotor rack (TOMY DIGITAL BIOLOGY, model: AR015-24 )
  13. Electrophoresis apparatus (Bio-Rad Laboratories, model: 1658033FC )
  14. Flat scanner (Epson, model: Epson Perfection V37 )
  15. Gel drying equipment (Vision Scientific, model: ME-2B-S )
  16. -80 °C freezer (NuAire, model: NU-6625D36 )
  17. Water bath (VISION SCIENTIFIC, model: VS-1205SW1 )
  18. Thermo shaker (BIOAND, model: MSC100 )
  19. Solid tapered microtip (Sonics & Materials, catalog number: 630-0418 )

Procedure

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版权信息

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

如何引用

Moon, S., Kim, J. and Jho, E. (2017). In vitro NLK Kinase Assay. Bio-protocol 7(21): e2593. DOI: 10.21769/BioProtoc.2593.

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癌症生物学 > 增殖信号转导 > 生物化学试验 > 蛋白质分析

生物化学 > 蛋白质 > 活性

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