Hiprep 16 60 sephacryl s 200 high resolution column

Cleavage assays used purified GelE. Purified GelE was obtained from ▽sprE as described previously [27 (link),38 (link),39 (link)]. Briefly, cells were grow to stationary phase and the culture medium was filtered with 0.45 and 0.22 μm pore-size filters. Proteins were precipitated overnight at 4°C with the addition of ammonium sulfate crystals to a saturation of 60%. After centrifugation, the protein pellet was dialyzed overnight 4°C in 50 mM Tris-HCl-1 mM CaCl₂, pH 7.8. A second concentration step was carried out using a Millipore stirred-cell concentrator with a 10,000-molecular weight cutoff membrane. Sample was then applied to a HiPRep 16/60 Sephacryl S-200 high-resolution column (GE Healthcare) and tested for gelatinase activity via gelatin agar plates [40 (link)].
For rAtlA cleavage, reaction mixtures containing 3 μg of rAtlA and various amounts of purified GelE (ranging from 0 to 1.5 μg) in 0.1 ml of PBS were incubated for 30 minutes at 37°C. The enzyme activity was halted by the addition of SDS-PAGE sample buffer containing 2-mercaptoethanol followed by boiling. Purified GelE (1.5 μg) and rAtlA (3.0 μg) were diluted in 0.1 ml of PBS and incubated similarly to serve as controls. Samples were applied onto SDS-PAGE gradient gels (4 to 20%) (NuSep, Inc.), and developed with Coomassie blue protein stain (Sigma).

Two mg of HEK293 cell cytoplasmic extract was filtered through a 0.2 µ low protein binding filter (Millipore) and then loaded at 4°C into a calibrated HiPrep 16/60 Sephacryl S-200 High Resolution column (GE) in 15 mM Tris-HCl (pH 7.5), 150 mM NaCl. The column was developed in the same buffer at a flow rate of 0.5 ml/min, and starting from the void volume (elution volume 37 ml) 0.5 ml fractions were collected up to an elution volume of 62 ml in order to separate monomeric proteins as small as 29 kDa. 250 µl from each fraction was TCA precipitated and analyzed by Western blotting with anti-CE and anti-Nck1 antibodies. The remaining 250 µl of the four fractions indicated with a box in Figure 3B were pooled and immunoprecipitated with control IgG or anti-Nck1 antibody and Dynabeads protein G. The recovered proteins were analyzed by Western blotting with anti-CE and anti-Nck1 antibodies.