Supporting Information

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1 Supporting Information Ni/NiO Core/shell Nanoparticles for Selective Binding and Magnetic Separation of Histidine-Tagged Proteins In Su Lee,, No Hyun Lee,, Jongnam Park,, Byung Hyo Kim,, Yong-Weon Yi, Taeuk Kim, Tae Kook Kim, In Hwan Lee, Seung R. Paik, and Taeghwan Hyeon*,, National Creative Research Initiative Center for Oxide Nanocrystalline Materials and School of Chemical and Biological Engineering, Seoul National University, Seoul , Korea, and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon , Korea. Preparation of Ni/NiO nanoparticles General consideration. Reaction solvents including hexane, toluene, ethanol, and acetone were distilled through standard procedure. Bis(cyclopentadienyl)nickel (Nickelocene, 99 %, Strem), oleylamine (70 %, Aldrich), and tioctylphosphine (TOP, 90 %, Aldrich) were used as purchased without further purification. Trioctylphosphine oxide (TOPO, 90 %, Aldrich) was purified by vacuum distillation before use. Histagged GFP and normal mouse IgG 1 conjugated by PE-Cy5 were purchased from Santa Cruz Biotechnology and Upstate, respectively. Photoluminescnet spectra were collected on a Perkin-Elmer LS 50B with an excitation wavelength of 400nm (for GFP) and S1

2 630nm (for Cy5). Transmission electron microscopy (TEM) was conducted with JEOL JEM Fluorescent image of Ni/NiO nanopartcles capturing GFP was obtained using confocal laser scanning microscopy of Carl Zeiss LSM-510 at National center for inter-university research facility at the Seoul National University. Magnetic properties of nanoparticles were measured using superconducting quantum interference device (SQUID) magnetometer (Quantum Design, MPMS5XL), which is equipped with a 5 T superconducting magnet. Synthesis of Ni/NiO naoparticle. Ni-oleylamine complex was prepared by reacting Ni(acac) 2 (0.2 g) and oleylamine (2.0 ml) with heating under an Ar atmosphere. The Nioleylamine complex solution was injected into the mixture solution of trioctylphosphine oxide (TOPO, 5.0 g) and trioctylphosphine (0.3 ml) and slowly heated up to 250 C. The resulting solution was aged for 30 min at 250 C, and was then cooled to room temperature. The nanoparticles were precipitated by adding excess ethanol to the solution. The precipitated nanoparticles were retrieved by centrifugation. The nanoparticles were dispersed in hexane and aged for several days under air for the oxidation. Resulting Ni/NiO nanoparticles were obtained as black solids by the addition of acetone and subsequent centrifugation. Ligand exchange of Ni/NiO naoparticle. Ni/NiO nanoparticles (200 mg) were dispersed into chloroform solution containing imidazole (0.5 g/ml, 5 ml) and refluxed for 6 hr. After cooling to room temperature, a powder form of imidazole stabilized Ni/NiO nanoparticles were obtained by addition of n-hexane, centrifugation, and washing with EtOH. S2

3 Investigation of binding and separation of His-tagged proteins Reaction of Ni/NiO nanoparticles with proteins. Ni/NiO nanoparticles (50 μg) were added to protein solution containing His-tagged GFP or normal mouse IgG 1 conjugated by PE-Cy5 in PBS (30 μg/ml, 250 μl) and incubated with shaking for 30 min. Ni/NiO nanoparticles were isolated from the supernatant by using a magnet, redispersed into imidazole solution (0.1 g/ml, 250 μl), and incubated with shaking for 30 min to release proteins captured by the nanoparticles. Experiment for the separation of His-tagged proteins from untagged proteins were carried out with the mixture solution of Histagged GFP and normal mouse IgG 1 conjugated by PE-Cy5 (30 μg/ml for each). Expression and purification of His-tagged proteins from cell lysate. The expression plasmid, encoding the Fc binding domain of protein G fused with N-terminal His-tag (His-FcBD), was a kind gift from Dr. C. S. Shim (ELPIS Biotech Co., Ltd., Daejeon, Korea). A 12 ml culture of BL21(DE3) cells expressing His-FcBD was grown to OD 600 ~0.5, induced with 0.2 mm IPTG for 2 hrs at 37 C, and then pelleted by centrifugation, resuspended in 1 ml of phosphate-buffered saline (PBS). After disrupting the cells by sonication, soluble fractions were obtained by centrifugation at 4 C with 12,000 rpm for 20 min. The soluble fractions were incubated with Ni/NiO nanoparticles at room temperature for 10 min with shaking. After two times wash by PBS containing with 10 mm imidazole, His-FcBD proteins were eluted with PBS supplemented with 500 and 1000 mm of imidazole, respectively. Proteins were resolve on 8% GradiGel II Gradient PAGE Analysis Kit (ELPIS Biotech Co., Ltd.). The gel was stained with Coomassie blue. (Figure S3) S3

4 Figure S1. Confocal laser microscopy image obtained from His-tagged GFP protein bound Ni/NiO nanoparticles in a powder form. Figure S2. Fluorescence spectra showing the change of emission intensity of the solutions in Figure 3a. S4

5 Figure S3. Purification of His-FcBD from E. coli using Ni/NiO nanoparticles. Fractions were collected from each of the purification steps and analyzed by SDS-PAGE. Lane M, molecular weight markers; Lane 1: crude lysate before induction of protein; Lane 2: crude lysate after induction of protein; Lane 3: insoluble fraction. Lane 4: soluble fraction; Lane 5: flow-through of the lysate after incubation with Ni/NiO nanoparticles; Lane 6-7: two washes with PBS containing 10 mm imidazole; Lane 8-9: His-FcBD eluted from the nanoparticles with PBS containing 500 mm imidazole; Lane 10-11: His- FcBD eluted from nanoparticles with PNS containing 1000 mm imidazole; Lane 12: Proteins released from the nanoparticles by boiling after imidazole elution. S5