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Example sentences for: gst-t

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GST-T7-Cdc14 (RDB 872) and GST-Cdc14 (RDB 1183) were expressed in E. coli BL21(DE3) pLysS cells, and purified on glutathione-agarose beads (Sigma) as described previously [ 33 ] . His6-T7-Net1(1-341) (Net1N, RDB1153) [ 11 ] was expressed in E. coli BL21(DE3) pLysS cells, and purified on Ni-NTA agarose beads (Qiagen) as described previously for p28 Kix1 [ 34 ] . Purification of His6-Plx1, His6-Plx1(N172), and His6-HA-Cdc5 from baculovirus-infected insect cells followed an earlier protocol [ 24 ] . The concentrations of purified proteins were in the ranges of: Net1N~0.
His6-T7-Net1N/GST-T7-Cdc14 complex was assembled on anti-GST beads, and treated with either active or N172A kinase-inactive Plx1 (Polo-like kinase from Xenopus ; [ 24 ] ) in the presence of ATP.
For experiments in Figure 5, all four reactions consisted of 125 ng GST-T7-Cdc14 adsorbed to 20 μl anti-GST beads, 200 ng His6-T7-Net1N, 1 μl Plx1, 20 μl kinase buffer containing ATP, and the ATP depletion system (see below).
The beads were incubated with 250 ng of GST-T7-Cdc14 in 100 μl of HEBD for 1 hr, followed by the same sequence of washes.