Hiprep 16 60 sephacryl s 300 hr

αA-Crystallin with a His tag attached to the carboxy terminal end was also purified using a modification of the above-described method. The lysis buffer used in purifying αA crystallin was 50 mM KH₂PO₄/K₂HPO₄ pH 8.0, 300 mM KCl, 1 mM EDTA (buffer C) with a protease inhibitor mix (Roche Diagnostics; catalog no. 11836153001). Sonication was done as described above for the wild-type protein (6 cycles, each cycle comprising a 10 s pulse on/50 s pulse off) and the centrifuged cell lysate was loaded onto a HisTrap HP (GE Healthcare; catalog no. 17524801) column equilibrated with buffer C at a flow rate of 1.0 mL/min. Five column volumes of buffer C was passed through the column to wash off the unbound proteins completely, then a linear gradient of buffer D (50 mM KH₂PO₄/K₂HPO₄ pH 8.0, 300 mM KCl, 1 mM EDTA, and 0.5 M imidazole) was applied, and fractions were collected. Because the His tag had no effect on the αA-crystallin function when compared with the untagged protein, it was not removed before use in assays. αA samples were further purified by size exclusion chromatography as above using a HiPrep 16/60 Sephacryl S-300 HR (GE Healthcare; catalog no.17116701). Four independent preparations for each batch of αA-crystallin gave an average mass of 20731.06 ± 1 using ESI-LC/MS, in agreement with the predicted monomer mass.