In vitro Dephosphorylation Assay of c-Myc
c-Myc体外去磷酸化实验
发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2011 浏览次数: 8616
评审: HongLok LungGabriel O. FerreroJianwei Sun
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参见作者原研究论文
The authors used this protocol in:
Jan 2016
Abstract
This protocol describes experimental procedures for in vitro dephosphorylation assay of human protein c-Myc. This protocol can be adapted to detect phosphatase activity of other Ser/Thr phosphatases.
Keywords: Dephosphorylation (去磷酸化)
c-Myc (c-Myc)
SCP1 (SCP1)
Background
Carboxy-terminal domain RNA polymerase II polypeptide A small phosphatase 1 (SCP1, also known as CTDSP1 or NLI-IF) belongs to the FCP/SCP phosphatase family and was originally reported to dephosphorylate the C-terminal domain (CTD) of RNA polymerase II (Yeo et al., 2003). Smad2, 3 (Wrighton et al., 2006), Snail (Wu et al., 2009), PML (Lin et al., 2014), and c-Myc (Wang et al., 2016) have also been identified as substrates of SCP1.
Materials and Reagents
- 1.5 ml Eppendorf tubes
- 50 ml tube
- 100 mm dish
- E. coli cells
- HKE293T cell
- HA-c-Myc plasmid
- LB medium
- IPTG
- Glutathione sepharose 4B (GE Healthcare, catalog number: 17075601 )
- Glycerol (Sigma-Aldrich, catalog number: G5516 )
- PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 23225 )
- Lipofectamine 2000
- c-Myc antibody (Abcam, catalog number: ab32072 )
- Protein A/G beads (Santa Cruz Biotechnology, catalog number: sc-2003 )
- BSA
- Coomassie blue R250 (Sigma-Aldrich, catalog number: 27816 )
- c-Myc Ser62 phosphorylation antibody ([Abnova, catalog number: MAB6763 ] or [Abcam, catalog number: ab78318 ])
- Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: 221465 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S7653 )
- Potassium chloride (KCl)
- Disodium hydrogen phosphate (Na2HPO4)
- Potassium phosphate monobasic (KH2PO4)
- Tris base (C4H11NO3) (Sigma-Aldrich, catalog number: T1503 )
- EDTA (Sigma-Aldrich, catalog number: E5134 )
- Triton X-100 (Sigma-Aldrich, catalog number: X100 )
- Phenylmethanesμlfonyl fluoride (PMSF) (C7H7FO2S) (Sigma-Aldrich, catalog number: P7626 )
- cOmplete protease inhibitor cocktail (Roche Diagnostics, catalog number: 04693116001 )
- Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D0632 )
- PhosSTOP tablet (Roche Diagnostics, catalog number: 04906845001 )
- β-mercaptoethanol
- Bromophenol blue
- Sodium dodecyl sulfate (SDS)
- Magnesium chloride (MgCl2) (Sigma-Aldrich, catalog number: 449172 )
- L-Glutathione reduced (Sigma-Aldrich, catalog number: G4251 )
- Phosphate-buffered saline (PBS) (see Recipes)
- IP lysis buffer (see Recipes)
- Phosphatase reaction buffer (see Recipes)
- 5x SDS loading dye (see Recipes)
- GST elution buffer (see Recipes)
Equipment
- 250 ml flask
- Shaker
- Cell scraper
- Incubator
- Centrifuges
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Liao, P., Wang, W. and Ge, X. (2017). In vitro Dephosphorylation Assay of c-Myc. Bio-protocol 7(2): e2011. DOI: 10.21769/BioProtoc.2011.
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分类
癌症生物学 > 癌症生物化学 > 蛋白质
生物化学 > 蛋白质 > 活性
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