2018
DOI: 10.1021/acs.analchem.8b02667
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Integrated and Quantitative Proteomic Approach for Charting Temporal and Endogenous Protein Complexes

Abstract: Proteins often assemble into multiprotein complexes for carrying out their biological functions. Affinity purification combined with mass spectrometry (AP-MS) is a method of choice for unbiasedly charting protein complexes. Typically, genetically tagged bait protein and associated proteins are immunoprecipitated from cell lysate and subjected to in-gel or on-bead digestion for MS analysis. However, the sample preparation procedures are often time-consuming and skipping reduction and alkylation steps results in… Show more

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Cited by 16 publications
(34 citation statements)
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“…RAR3G‐APEX2‐FLAG plasmid (from the laboratory of Dr Ruijun Tian in Sust, Shenzhen, China) expresses APEX2‐flag, containing Tet‐on doxycycline‐inducible expression vector, and could be used for lentivirus infection and packaging for the generation of stable cell line. To construct APEX2 labeling plasmid RAR3G‐APEX2‐FLAG‐λN, a 66‐bp λN (BoxB binding protein) sequence was codon‐optimized for mammalian cell expression and synthesized, introduced by homologous recombination according to the manufacturer's instructions (ClonExpress II One Step Cloning Kit; Vazyme).…”
Section: Methodsmentioning
confidence: 99%
“…RAR3G‐APEX2‐FLAG plasmid (from the laboratory of Dr Ruijun Tian in Sust, Shenzhen, China) expresses APEX2‐flag, containing Tet‐on doxycycline‐inducible expression vector, and could be used for lentivirus infection and packaging for the generation of stable cell line. To construct APEX2 labeling plasmid RAR3G‐APEX2‐FLAG‐λN, a 66‐bp λN (BoxB binding protein) sequence was codon‐optimized for mammalian cell expression and synthesized, introduced by homologous recombination according to the manufacturer's instructions (ClonExpress II One Step Cloning Kit; Vazyme).…”
Section: Methodsmentioning
confidence: 99%
“…mCdx2 and rCdx2 overexpression (OE) vectors were constructed by replacing APEX2 with mCdx2 or rCdx2 in the RAR3G -APEX2 -FLAG plasmid [41] based on the homologous recombination technology (Vazyme C112). The mutated Cdx2 plasmids were modified from the rCdx2 plasmid based on ClonExpress rapid cloning technology according to the manufacturer's instructions (Vazyme C215).…”
Section: Plasmid Vector Constructionmentioning
confidence: 99%
“…Plasmid vector construction mCdx2 and rCdx2 overexpression (OE) vectors were constructed by replacing APEX2 with mCdx2 or rCdx2 in the RAR3G-APEX2-FLAG plasmid [41] based on the homologous recombination technology (Vazyme C112). The mutated Cdx2 plasmids were modi ed from the rCdx2 plasmid based on ClonExpress rapid cloning technology according to the manufacturer's instructions (Vazyme C215).…”
Section: Cell Culturementioning
confidence: 99%