User:Tamra L. Fisher/Notebook/Experimental Biological Chem/2012/02/01

AU Biomaterials Design Lab

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Objective Prepare media for GFP expression next week. Using affinity chromatography, purify the MBP protein with a His-tag, that had previously been attempted to be purified by other members of the class. Description Preparation of Media for Expression Combine 25g LB and 1L of distilled water in a 2.8L flask. (2 flasks) Combine 0.875g LB, 0.7g agar, and 35mL distilled water in a 250mL flask. Autoclave the flasks (liquid cycle). Add 35μL of 100mg/mL ampicillin to the mixture in the 250mL flask when cooled to ~60°C. Pour the mixture in the 250mL flask into a sterile petri plate. Filtering the MBP with His-tag Groups 2-5 in the previous semester attempted to purify the MBP with His-tag, but did not seem to get much protein. Their purification products were saved and stored at 4°C. the groups saved the fractions from their purification in various "peaks" based on the data they observed from the FPLC. Peaks 3 and 4 will be repurified by our group using affinity chromatography. Over the time they were stored it appeared that bacteria began to grow in some of the collections. Using a 60mL syringe, the fractions were pulled into the syringe and then pushed through a 0.2μM filter. The fractions from the different groups were combined, but the peaks were kept separate. Purifying "Peak 3" The filtered peak 3 fractions (~100mL) were pumped over the regenerated amylose resin column at 1.5mL/min. Wash the column with 100mL of column buffer (20mM Tris-HCl (pH 7.4), 200mM NaCl, and 1mM EDTA) at 1.5mL/min. Elute the protein from the column with 100mL 10mM maltose in column buffer. 10mM maltose(100mL) = 0.36g maltose + column buffer Streaking out E.coli with MBP Obtain the glycerol stock (pMXB10) (Stock #115) from the -80°C freezer and store on dry ice. Using a sterile wooden stick scrape the top of the glycerol stock. Streak the wooden stick across the petri plate in a zig-zag pattern. Store the glycerol stock in the -80°C freezer again.