Ni nitrilotriacetic acid nta agarose beads

Plasmids pET15b-OprF/I, expressing 6×His-OprF/I, or pIZ2338, expressing GST-PcrV, were transformed into E. coli BL21(DE3). Bacteria were grown in LB with ampicillin at 37°C. At mid-exponential-growth phase, 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was added to induce expression, and incubation was pursued for 4 h. The cells were harvested by centrifugation, and the cell pellet was resuspended in NP-40 lysis buffer (64 (link)). A commercial mixture of protease inhibitors (Sigma-Aldrich) was added to the suspension, and lysis was performed by sonication. The cell lysate was centrifuged at 15,000 × g for 30 min at 4°C, and the fusion proteins were isolated from the supernatants by affinity chromatography with Ni-nitrilotriacetic acid (NTA) agarose beads (Qiagen) or glutathione agarose beads (Sigma-Aldrich) according to the manufacturers’ protocols.

His₆-tagged PML was isolated from CHO-PML cells lysed with lysis buffer (6 M guanidium HCl, 10 mM Tris, 100 mM sodium phosphate buffer [pH 8.0], 5 mM β-mercaptoethanol, 1 mM imidazole). The cell lysates were sonicated and centrifuged for 15 min at 16,000 × g, and the corresponding supernatants were incubated overnight at 4°C with 250 μl of packed Ni-nitrilotriacetic acid (NTA) agarose beads (Qiagen) prewashed in lysis buffer. After incubation, the beads were washed once in lysis buffer, once in wash buffer, pH 8.0 (8 M urea, 10 mM Tris, 100 mM sodium phosphate buffer [pH 8.0], 0.1% Triton X-100, 5 mM β-mercaptoethanol), and three times in wash buffer, pH 6.3 (8 M urea, 10 mM Tris, 100 mM sodium phosphate buffer [pH 6.3], 0.1% Triton X-100, 5 mM β-mercaptoethanol, 10 mM imidazole). His₆-tagged PML proteins were then eluted from the beads using elution buffer (200 mM imidazole, 5% SDS, 150 mM Tris-HCl [pH 6.7], 30% glycerol, 720 mM β-mercaptoethanol, 0.0025% bromophenol blue).

Sodium dodecyl sulfate (SDS), alginate (A7003), phenylmethylsulfonylfluoride (PMSF), imidazole, and dithiothreitol (DTT) were obtained from Sigma Aldrich (St. Louis, MO), whereas cloned pfu DNA polymerase was purchased from Stratagene (La Jolla, CA). DpnI endonuclease, protein A-horseradish peroxidase (HRP), polyvinylidene fluoride, and pET28A(+) expression vector were obtained from New England BioLabs (Ipswich, MA), BD Transduction Laboratories (BD Biosciences, Franklin Lakes, NJ), EMD Millipore (Billerica, MA), and Invitrogen (Waltham, MA), respectively. The QIAprep Spin Plasmid Miniprep Kit and Ni-nitrilotriacetic acid (NTA) agarose beads were purchased from Qiagen (Venlo, Netherlands). The ECL Plus Western blotting detection system RPN 2132 was obtained from Amersham Life Science (GE Healthcare, Cleveland, OH). The oligonucleotide primers were synthesized by Bioneer (Daejeon, Korea) and the analysis facility of Chosun University. Acetylated alginate was purified from Pseudomonas as previously described, using an alginate-overproducer, P. alkylphenolia E1 (pAlgG). Purified acetylated alginate was converted to a Na-form by treating with 50 mM ethylene diamine tetraacetic acid (EDTA), and dialysis using 0.5 M NaCl and double distilled water [41 ].