cation exchange binding buffer  (GE Healthcare)

Journal: Biochemical Journal

Article Title: Efficient overexpression and purification of severe acute respiratory syndrome coronavirus 2 nucleocapsid proteins in Escherichia coli

doi: 10.1042/BCJ20240019

Figure Lengend Snippet: SDS–PAGE (10%) analysis of samples from example purification. Molecular weight markers (MW, Bio-Rad (Cat. 161-0362)) are shown for each panel. Left panel shows total SDS-cell lysates of uninduced and induced E. coli BL21 carrying plasmid pT5P_MHTOmicronNcap (Lanes 1 and 2 respectively). The soluble fraction from induced cells after lysis, sonication and centrifugation (Lane 3). Pellet from 5% to 17% ammonium sulfate precipitation (Lane 4) and corresponding supernatant (Lane 5). The ammonium sulfate-protein pellet was resuspended in HisTrap loading buffer, centrifuged to separate into insoluble material (Lane 6) and the soluble fraction (Lane 7) and loaded on a HisTrap column (Lane 8). Lane 9 shows proteins passing through the HisTrap column. Pooled fractions eluted from the HisTrap column (Lane 10) were diluted with cation exchange loading buffer and loaded on SP Sepharose column (Lane 11). Flow through from SP column (Lane 12). Peak fractions from linear gradient elution (Lanes 13 and 14). Right panel shows samples of purified 6His-tagged; Ncap Omicron (Lane 1); Ncap Delta (Lane 2); Ncap Alpha (Lane 3); Ncap 203/204 (Lane 4); Ncap (Lane 5) as well as untagged Ncap (Lane 6).

Article Snippet: The purest fractions were diluted 10-fold into cation exchange loading buffer (20 mM HEPES pH 8, 10 mM NaCl, 1 mM EDTA, 5% v/v glycerol) for all protein except Delta, in which case the buffers were adjusted to 1 mM in DTT to keep the cysteine residues reduced before chromatography on a 20 ml HiPrep SP FF 16/10 column (Cytiva).

Techniques: SDS Page, Purification, Molecular Weight, Plasmid Preparation, Lysis, Sonication, Centrifugation