Read the methods section below from “E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1)

Read the methods section below from “E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1).” (Vichi, A. et. Al., 2005. Proc Natl Acad Sci U S A. Feb 8;102(6):1945-50). Fusion Protein Synthesis and Purification. Single colonies of Escherichia coli XL1 blue or BL21(DE3) gold (Stratagene) containing plasmids with inserts encoding GST-tagged ARD1 or related proteins were added to 5 ml of Luria-Bertani broth containing ampicillin at 100 ?g/ml. After incubation overnight at 37°C with shaking, the culture was added to 500 ml of the same medium and incubated at 37°C with shaking until A600 = 0.6. Isopropyl-?-D-thiogalactoside was added (0.2 mM final concentration), and after incubation at 37°C for 4 h, cells were sedimented by centrifugation (5,000 X g for 10 min) and stored at -20°C. Recombinant proteins were purified essentially as described by Frangioni and Neel (20). Briefly, cells were dispersed in 20 ml of STE buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8) containing lysozyme at 1mg/ml. After incubation for 1 h on ice and the addition of 1% Triton X-100, cells were sonified and incubated (1 h at room temperature, shaking) with DNase I (Roche Applied Science) at 44 units/ml. Inclusion bodies were collected by centrifugation (15,000 X g for 15 min) and dispersed in 20 ml of STE. After the addition of 1 ml of 20% Sarkosyl and intermittent mixing (vortex mixer, 5 s every 30 s) for 5-10 min,1 ml of 20% Triton X-100 was added, insoluble material was discarded after centrifugation (15,000X g for 10 min at 4°C), and the clear supernatant was incubated (2 h at 4°C) with 0.25 ml of reduced glutathione-Sepharose. The mixture was transferred to a column, and beads were washed three times with 10 ml of STE buffer. Bound proteins were eluted with three 0.5-ml portions of 10 mM reduced glutathione in 50 mM Tris/HCl (pH 8) and concentrated by using Microcon centrifugal filter devices (10,000 or 100,000 molecular weight cut-off; Millipore). The protein concentration was determined by the Bradford method (21). Purity assessed by silver staining after SDS/PAGE was >90%. After addition of propylene glycol (35% final concentration), protein (0.1-1 mg/ml) was stored in small portions at -20°C. For ubiquitinylation assays, two different preparations of GST- or His-ARD1 protein were used.

1. How do you prepare the 20 ml of STE buffer the authors used to dissolve the cells (show work)? ____________ Tris ___________ NaCl __________ EDTA Method: ___________________________________________________________

2. What is the purpose of EDTA in this buffer? ___________________________________________________________

3. What three different techniques did the authors use to lyse these bacterial cells? __________________________ __________________________ __________________________

4. What is DNase I? _______________________________ Why is it listed in units/ml and not mg/ml? _______________________________

5. What type of chromatography was used to purify the recombinant proteins? _______________________________ What was used to elute the protein from the beads?