Densitometric analysis of the TRAP gel allowed measurements of relative telomerase

the starter culture was used to inoculate 1 L of 2xTY broth supplemented with kanamycin. The cells were grown to an OD600 = 0.5. Expression of Ariflo MalE-VirF was induced with the addition of arabinose and the culture continued to shake at 37 for an additional 5 hours. Cells were then harvested via centrifugation and were stored overnight at -20. The next day the cells were resuspended in 20 mL of amylose resin binding buffer supplemented with phenylmethylsulfonyl fluoride and 20 ��L of lysonase bioprocessing reagent. Cells were slowly stirred for 10 minutes at room temperature and were then immediately placed on ice and kept on ice or at 4 for the remainder of the procedure. Cells were lysed via sonication utilizing a ultrasonic XL2020 sonicator. Following sonication, cellular debris were removed via centrifugation. The resultant supernatant was then applied to a 10 mL NKL 22 structure column of amylose resin by gravity flow. Before addition of the supernatant the column was washed with 8 column volumes of amylose resin binding buffer. Following addition of the supernatant, the column was washed with 12 column volumes of amylose resin binding buffer. MalE-VirF was eluted from the column in 1 mL fractions of amylose resin elution buffer and 10 mM maltose). Fractions were analyzed by SDS-PAGE. Fractions containing MalEVirF were pooled, concentrated to approximately 6.5 mg/mL using Amicon Ultra-15 centrifugal units , and stored in liquid nitrogen for future use. Analytical gel filtration chromatography was used to determine the oligomeric state of purified MalE-VirF. Briefly, MalE-VirF was applied to a Superose 12 column, which was equilibrated with amylose resin binding buffer using an AKTA FPLC system. The sample was run through the column at a flow rate of 0.75 mL/min using amylose resin binding buffer. Eluted proteins were detected spectrophotometrically at 280 nm. The oligomeric state of MalE-VirF was determined by comparison to a previously generated 4-point molecular weight calibration curve specific to the Superose 12 column. DNA probes were utilized in both EMSA, FP, and FID assays. The sequences of the oligonucleotides were based on previous studies and are listed in Table