c-Myc体外去磷酸化实验

Peng Liao; Weichao Wang; Xin Ge

doi:10.21769/BioProtoc.2011

Improve Research Reproducibility A Bio-protocol resource

提交稿件
订阅
CN
- EN - English
- CN - 中文

该实验方案已做出更正，查看更正通知。

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: 8400

评审: HongLok LungGabriel O. FerreroJianwei Sun

下载 PDF

Q&A

引用

Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Oncogene, featuring study using the protocol.

Jan 2016

实验方案合集

Cell Imaging - A Special Collection for Cell Bio 2023

相关实验方案

基于Azo-Xylan及其变体的Endo-1,4-β-D-木聚糖酶活性检测方法

Luca Bombardi [...] Salvatore Fusco

2025年04月20日 935 阅读

基于体外激活方法的X-琥珀酸合酶活化及其在富马酸加烷基反应中的应用

Anshika Vats [...] Mary C. Andorfer

2025年06月20日 1484 阅读

测定丙酮酸激酶动力学的优化酶偶联分光光度法

Saurabh Upadhyay

2025年08月20日 846 阅读

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)