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Published in final edited form as: Genes Cells. 2016 Jan 13;21(2):163–184. doi: 10.1111/gtc.12334

1L cultures of cells expressing RecG-his, PriA-biotin and profinity-SSB were grown to early log phase in LB+ antibiotics and biotin, IPTG added to 200 µM and growth continued until early stationary phase. Cells were harvested by centrifugation, lysed and the cleared cell lysate applied to a 5ml nickel column as described in the Experimental Procedures. Proteins were then eluted using an imidazole gradient following extensive washing to remove unbound proteins. Pooled fractions were applied to the profinity column, washed and cleavage allowed to occur overnight at 4°C. The next day, proteins were eluted using elution buffer as described by the manufacturer (Biorad). (A), scheme of the triple-tagged complex purification. (B), SDS-PAGE of relevant fractions from the purification. (C), blots from PAGE gels of the same fractions probed with anti-6x–his tag antibody (left) or streptavidin (right). Key: M, marker; Ni, nickel pool, PF, profinity flow through and PE, proteins eluted from the profinity column. (D), SDS-PAGE criterion gel lanes of fractions eluted from a nickel and gel filtration column. (E), blots of relevant lanes of the RecG-his/PriA-biotin purification. Key: M, marker; CCL, cleared cell lysate; FT, flow through; Ni, nickel pool and GF, peak fraction from the gel filtration column.

1L cultures of cells expressing RecG-his, PriA-biotin and profinity-SSB were grown to early log phase in LB+ antibiotics and biotin, IPTG added to 200 µM and growth continued until early stationary phase. Cells were harvested by centrifugation, lysed and the cleared cell lysate applied to a 5ml nickel column as described in the Experimental Procedures. Proteins were then eluted using an imidazole gradient following extensive washing to remove unbound proteins. Pooled fractions were applied to the profinity column, washed and cleavage allowed to occur overnight at 4°C. The next day, proteins were eluted using elution buffer as described by the manufacturer (Biorad). (A), scheme of the triple-tagged complex purification. (B), SDS-PAGE of relevant fractions from the purification. (C), blots from PAGE gels of the same fractions probed with anti-6x–his tag antibody (left) or streptavidin (right). Key: M, marker; Ni, nickel pool, PF, profinity flow through and PE, proteins eluted from the profinity column. (D), SDS-PAGE criterion gel lanes of fractions eluted from a nickel and gel filtration column. (E), blots of relevant lanes of the RecG-his/PriA-biotin purification. Key: M, marker; CCL, cleared cell lysate; FT, flow through; Ni, nickel pool and GF, peak fraction from the gel filtration column.