Amicon ultra 15 centrifugal unit

6xHis-MBP-HRV3Csite-HA-Cyclin A protein was expressed using a modified pET plasmid vector in E. coli.^{43 (link)}. The protein was purified using Ni-sepharose beads (GE Healthcare). The protein was incubated with 6xHis-HRV3C protease at 4 °C overnight to cleave off the 6xHis-MBP fragment from the CycA protein. The cleaved product, HA-CycA, was further purified by passing through Ni-sepharose, buffer-exchange, and concentration using Amicon-Ultra-15 centrifugal unit (EMD Millipore). The purified HA-CycA protein in buffer (20 mM Hepes-NaOH [pH 8.0], 500 mM NaCl, 0.25 mM TCEP, and 50% glycerol) was flash-frozen in liquid nitrogen and stored at −80 °C until use.
Hand-dissected stage 14 oocytes were homogenized in lysis buffer (30 mM Hepes-KOH [pH 7.4], 100 mM KOAc, 2 mM MgOAc), followed by protein concentration determination with BCA protein assay kit (Pierce). Then final 1 mM DTT was added to the lysates. About 200 ng of HA-Cyclin was incubated in the stage 14 oocyte lysates (about 540 ng total protein) in the presence of 1 mM ATP in 100 µl scale at 25 °C. Ten microliters of the reaction mixture was taken at each time point, mixed with 2× SDS PAGE loading buffer, and heated at 95 °C for 5 min for Western blot analysis. Half-life (t_1/2) was determined by curve fitting of an equation—Fraction remaining = (1/2)^(time/t_1/2)—using Igor Pro 6.31 (Wavematrix)^{56 (link)}.

Overnight culture of B. pseudomallei, Bp22, was inoculated into LB broth (Difco Laboratories, Detroit, Michigan) and incubated for 4 h at 37°C until an OD600 reading of 0.8–1.0. The bacteria was pelleted at 4000 g for 10 min and washed twice with 1X phosphate buffer saline (PBS), prior to reconstitution in 1X PBS containing 0.2 µg/ml leupeptin (Sigma-Aldrich, St Louis, MO), 0.2 µg/ml pepstatin A (Sigma-Aldrich, St Louis, MO) and 2.5 Kunitz units of DNase I (Sigma-Aldrich, St Louis, MO). To extract bacterial proteins, the suspension was loaded into lysing Matrix B tubes (MP Biochemicals, Solon, OH), and three rounds of homogenization was performed using Fastprep instrument (MP Biochemicals, Solon, OH) at a speed of 6 m/s for 30 s with a pause of 10–15 min in between for cooling. The bacterial lysate was centrifuged at 13000 g for 2 min, and passed through 0.22 µm filter unit (Merck Millipore, Billerica, MA) to remove any live bacteria. The supernatant was further purified and concentrated using Amicon Ultra-15 centrifugal units with 10 kDa nominal molecular weight limit (NMWL) membrane (Merck Millipore, Billerica, MA). The final purified Bp antigen were recovered in 1XPBS, quantified by BCA protein assay kit (Pierce Biotechnology, Rockford, IL) and used for further stimulation experiments.