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

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.

Automated cell disruption is a reliable and effective method of isolating RNA from fresh snap-frozen normal and malignant oral mucosa samples

This study compared automated vs. manual tissue grinding in terms of RNA yield obtained from oral mucosa biopsies.: A total of 20 patients undergoing uvulectomy for sleep-related disorders and 10 patients undergoing biopsy for head and neck squamous cell carcinoma were enrolled in the study. Samples were collected, snap-frozen in liquid nitrogen, and divided into two parts of similar weight. Sample grinding was performed on one sample from each pair, either manually or using an automated cell disruptor. The performance and efficacy of each homogenization approach was compared in terms of total RNA yield (spectrophotometry, fluorometry), mRNA quantity [densitometry of specific: Although spectrophotometry and fluorometry results were comparable for both homogenization methods,: Automated tissue homogenization appears to be a versatile, quick, and reliable method of cell disruption and is especially useful in the case of small malignant samples, which show unreliable results when processed by manual homogenization.Clin Chem Lab Med 2009;47:294–301.