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. 2016 Sep 8;12(5):3153–3160. doi: 10.3892/ol.2016.5106

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Copyright: © Tian et al.

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

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Figure 3. — Expression, purification and identification of the LP-15 RCC1 functional domain protein. (A) 12% SDS-PAGE of the cell lysate. Lanes 1–6, total cell lysate from DE3 induced using 1 mM IPTG for 6, 5, 4, 3, 2 and 1 h, respectively. pET-32a(+) served as a negative control. M, molecular weight marker. Lanes 7–12, total cell lysate from DE3 with pET-32a(+)-LP-15-RCC1 induced using 1 mM IPTG for 1, 2, 3, 4, 5 and 6 h, respectively. (B) Western blot of pET-32a(+)-LP-15-RCC1. Lane 1, total cell lysate from DE3 induced with 1 mM IPTG for 6 h. pET-32a(+) served as a negative control. Lanes 2–7, total cell lysate from DE3 cells with pET-32a(+)-LP-15-RCC1 induced using 1 mM IPTG for 1, 2, 3, 4, 5 and 6 h, respectively. (C) 12% SDS-PAGE of purified protein. M, protein molecular weight marker. Lane 1, total cell lysate after solubilization with buffer containing 8 M urea. Lane 2, liquid flow through from the Ni²⁺ column following addition of pET-32a(+)-LP-15-RCC1 sample. Lane 3, first phase eluent. Lane 4, second phase eluent. Lane 5, third phase eluent. Lane 6, fourth phase eluent. Lane 7, fifth phase eluent. Lane 8, sixth phase eluent. Lane 9, seventh phase eluent. RCC1, regulator of chromosome condensation 1; LP-15, Latcripin-15; IPTG, isopropyl β-D-thiogalactopyranoside; SDS-PAGE, sodium dodecyl sulfonate polyacrylate gel electrophoresis; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DE3, Rosetta-gami.