Biochemical Approaches Including the Design and Use of Strains Expressing Epitope-Tagged Proteins

被引：11

作者：

Couvillion, Mary T. ^{[1
]}

Collins, Kathleen ^{[1
]}

机构：

[1] Univ Calif Berkeley, Dept Mol & Cell Biol, Berkeley, CA 94720 USA

来源：

TETRAHYMENA THERMOPHILA | 2012年 / 109卷

关键词：

INDUCIBLE-REPRESSIBLE PROMOTER; DEPENDENT RNA-POLYMERASE; TETRAHYMENA-THERMOPHILA; WIDE IDENTIFICATION; GENE; TRANSFORMATION; BIOGENESIS; SEQUENCE; REPLACEMENT; DICER;

D O I：

10.1016/B978-0-12-385967-9.00012-8

中图分类号：

Q2 [细胞生物学];

学科分类号：

071009 ; 090102 ;

摘要：

Epitope tagging is a powerful approach used to enable investigations of a cellular component by elucidating its localization, interaction partners, and/or activity targets. Successful tag-based affinity purification yields a mixture of the molecule of interest, associated proteins and nucleic acids, and nonspecific background proteins and nucleic acids, many of which can depend on details of the protocol for enrichment. This chapter provides guidelines and considerations for designing an affinity purification experiment, beginning with construction of a strain expressing a tagged subunit. Common biochemical methods for detecting protein, RNA, and DNA in Tetrahymena thermophila are also discussed.

引用

页码：347 / 355

页数：9

共 29 条

[11] HIGH-FREQUENCY VECTOR-MEDIATED TRANSFORMATION AND GENE REPLACEMENT IN TETRAHYMENA [J].

GAERTIG, J ;

GU, L ;

HAL, B ;

GOROVSKY, MA .

NUCLEIC ACIDS RESEARCH, 1994, 22 (24) :5391-5398

[12] TRANSCRIPTIONAL PROPERTIES OF NUCLEOLI ISOLATED FROM TETRAHYMENA [J].

GOCKE, E ;

LEER, JC ;

NIELSEN, OF ;

WESTERGAARD, O .

NUCLEIC ACIDS RESEARCH, 1978, 5 (11) :3993-4006

[13]

Gorovsky M A, 1975, Methods Cell Biol, V9, P311, DOI 10.1016/S0091-679X(08)60080-1

[14] Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP [J].

Hafner, Markus ;

Landthaler, Markus ;

Burger, Lukas ;

Khorshid, Mohsen ;

Hausser, Jean ;

Berninger, Philipp ;

Rothballer, Andrea ;

Ascano, Manuel, Jr. ;

Jungkamp, Anna-Carina ;

Munschauer, Mathias ;

Ulrich, Alexander ;

Wardle, Greg S. ;

Dewell, Scott ;

Zavolan, Mihaela ;

Tuschl, Thomas .

CELL, 2010, 141 (01) :129-141

[15] Two classes of endogenous small RNAs in Tetrahymena thermophila [J].

Lee, SR ;

Collins, K .

GENES & DEVELOPMENT, 2006, 20 (01) :28-33

[16] Physical and functional coupling of RNA-dependent RNA polymerase and Dicer in the biogenesis of endogenous siRNAs [J].

Lee, Suzanne R. ;

Collins, Kathleen .

NATURE STRUCTURAL & MOLECULAR BIOLOGY, 2007, 14 (07) :604-610

[17] A single RNA-dependent RNA polymerase assembles with mutually exclusive nucleotidyl transferase subunits to direct different pathways of small RNA biogenesis [J].

Lee, Suzanne Rebecca ;

Talsky, Kristin Benjamin ;

Collins, Kathleen .

RNA, 2009, 15 (07) :1363-1374

[18] HITS-CLIP yields genome-wide insights into brain alternative RNA processing [J].

Licatalosi, Donny D. ;

Mele, Aldo ;

Fak, John J. ;

Ule, Jernej ;

Kayikci, Melis ;

Chi, Sung Wook ;

Clark, Tyson A. ;

Schweitzer, Anthony C. ;

Blume, John E. ;

Wang, Xuning ;

Darnell, Jennifer C. ;

Darnell, Robert B. .

NATURE, 2008, 456 (7221) :464-U22

[19] Germ line transcripts are processed by a Dicer-like protein that is essential for developmentally programmed genome rearrangements of Tetrahymena thermophila [J].

Malone, CD ;

Anderson, AM ;

Motl, JA ;

Rexer, CH ;

Chalker, DL .

MOLECULAR AND CELLULAR BIOLOGY, 2005, 25 (20) :9151-9164

[20] An RPA-Related Sequence-Specific DNA-Binding Subunit of Telomerase Holoenzyme Is Required for Elongation Processivity and Telomere Maintenance [J].

Min, Bosun ;

Collins, Kathleen .

MOLECULAR CELL, 2009, 36 (04) :609-619

← 1 2 3 →