PURIFICATION OF RAVE COMPLEX FROM Saccharomyces cerevisiae USING FLAG TAG-AFFINITY PURIFICATION METHOD

International Journal of Advanced Corporate Learning (iJAC)

View Publication Info
 
 
Field Value
 
Title PURIFICATION OF RAVE COMPLEX FROM Saccharomyces cerevisiae USING FLAG TAG-AFFINITY PURIFICATION METHOD
 
Creator Thanh Trung, Nguyen
Thi Tam, Pham
Thi Thu Thuy, Ta
Gu, Chunyin
Zhang, Zhenyu
 
Subject RAVE complex, Rav1, Rav2, Skp1, V-ATPase, Yeast, protein purification
 
Description RAVE (Regulator of the H+-ATPase of the Vacuolar and Endosomal membranes) is an            essential factor of assembly and reversible disassembly of V-ATPase. RAVE complex has three subunits, which are Rav1p, Rav2p and Skp1p. There are few studies on RAVE so it is very             important to study structure of RAVE complex to understand more about the regulation of the assembly and reassembly at V-ATPase. In this study the RAVE complex was purified by affinity purification by fussing FLAG tag to subunit Rav1p or Rav2p. Experimental process: yeast cells were incubated in 8 L YEPD medium at 30 oC, 200 rpm (OD600nm around of 3). Furthermore                   harvested cells were broken by a French pressure cell disruptor at 25,000 p.s.i in TBSE (50 mM Tris/Cl, 150 mM NaCl, 1 mM EDTA, pH 7.4) with 1 mm PMSF. The cell lysate was centrifuged at 20,000 xg for 20 minutes at 4 oC. Then, the supernatant, was achieved by centrifugation, and loaded onto a small column contained 1 ml of anti-FLAG M2 gel. After washing anti-FLAG column with TBSE, RAVE complex was eluted with TBSE containing 100 µg/ml FLAG               peptides. The results showed RAVE complex purification from strain with FLAG tag fused in                 C-terminus of Rav2 is better than RAVE complex purification from the yeast strain S. cerevisiae with FLAG tag fused in N-terminus of Rav1 or C-terminus of Rav1.
 
Publisher Publishing House for Science and Technology
 
Contributor
 
Date 2017-04-11
 
Type info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion


 
Format application/pdf
application/pdf
 
Identifier http://vjs.ac.vn/index.php/jst/article/view/9620
10.15625/0866-708X/51/5/9620
 
Source Vietnam Journal of Science and Technology; Vol 51, No 5 (2013); 543
Tạp chí Khoa học và Công nghệ; Vol 51, No 5 (2013); 543
2525-2518
2525-2518
 
Language vie
 
Relation http://vjs.ac.vn/index.php/jst/article/view/9620/7970
http://vjs.ac.vn/index.php/jst/article/view/9620/7971
 

Contact Us

The PKP Index is an initiative of the Public Knowledge Project.

For PKP Publishing Services please use the PKP|PS contact form.

For support with PKP software we encourage users to consult our wiki for documentation and search our support forums.

For any other correspondence feel free to contact us using the PKP contact form.

Find Us

Twitter

Copyright © 2015-2018 Simon Fraser University Library