c-Myc体外去磷酸化实验

Peng Liao; Weichao Wang; Xin Ge

doi:10.21769/BioProtoc.2011

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Peer-reviewed

In vitro Dephosphorylation Assay of c-Myc

c-Myc体外去磷酸化实验

Peng Liao

WW Weichao Wang

XG Xin Ge

发布: 2017年01月20日第7卷第2期 DOI: 10.21769/BioProtoc.2011 浏览次数: 8398

评审: HongLok LungGabriel O. FerreroJianwei Sun

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The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Jan 2016

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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 )
Pierce^TM BCA Protein Assay Kit (Thermo Fisher Scientific, Thermo Scientific^TM, 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)