Alkaline nuclease (AN) of the multiple-capsid nucleopolyhedrovirus (Accontain double-stranded (ds) circular DNA genomes of 100 to 180 kb, depending on the strain of disease (13). be directly involved in homologous recombination of the herpesvirus genome (R. Reuven, A. Staire, R. Myers, and S. Weller, submitted for publication). This observation suggested the Fagomine manufacture baculovirus homolog of herpesvirus AN and an unidentified DNA-binding protein may also participate in the baculovirus genome recombination. With this statement we describe further characterization of Ac9 (Sf9) cells were cultured in Sf900II serum-free medium (Invitrogen) with penicillin G (50 models/ml), streptomycin (50 g/ml; Whittaker Bioproducts), and fungizone (amphotericin B; 375 ng/ml) (Circulation Laboratories) as previously explained (12). Recombinant baculovirus vAcHISAN for overexpression of an AcDNA was labeled with [3H]thymidine (14). Purification of *AN/L3. Sf9 cells at a density of 1 1.5 106/ml in shaker flasks were infected with the recombinant baculovirus vAcHISAN at a multiplicity of infection of about 4 and incubated with shaking for 48 h at 28C. *AN/L3 was purified regularly from 100-ml ethnicities of infected cells by sequential liquid Fagomine manufacture chromatography on Ni-nitrilotriacetic acid (NTA)-agarose (Qiagen), DEAE-Toyopearl 650 (TosoHaas), and heparin-Sepharose CL-6B (Amersham Pharmacia Biotech) columns. The infected cells were pelleted by centrifugation for 5 min at 500 and resuspended in 8 ml of lysis buffer containing 50 mM Tris-HCl (pH 8.5), 200 mM KCl, 1% Nonidet P-40, 5 mM 2-mercaptoethanol, and a set of protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 M pepstatin, 5 g of leupeptin per ml, 5 g of aprotinin per ml, 2 g of E64 per ml). After extraction for 15 min at 4C on a rotating shaker, the planning was clarified by centrifugation at 30,000 for 30 Fagomine manufacture min. The supernatant was loaded onto a Ni-NTA-agarose column (0.8 ml), equilibrated with buffer A (20 mM Tris-HCl [pH 8.5], 0.5 M KCl, 10% [vol/vol] glycerol, 5 mM 2-mercaptoethanol, 20 mM imidazole). The column was washed successively with 10 ml of buffer A, 2 ml of buffer B (20 mM Tris-HCl [pH 8.5], 1 M KCl, 10% [vol/vol] glycerol, 5 mM 2-mercaptoethanol), 1 ml of buffer A, and finally with 2 ml of buffer C (20 mM Tris-HCl [pH 8.5], 75 mM KCl, 10% [vol/vol] glycerol, 5 mM 2-mercaptoethanol) containing 20 mM imidazole. Protein was eluted from your column with 4 ml Fagomine manufacture of buffer C containing 150 mM imidazole. The sample was loaded at a rate of 4 ml per h onto a DEAE-Toyopearl column (0.5 by 2.5 cm) equilibrated with buffer D (10 mM Tris-HCl [pH 7.5], 20% [vol/vol] glycerol, 1 mM dithiothreitol [DTT], 1 mM EDTA) containing 75 mM KCl. The column was washed successively with 1 ml of buffer D containing 75 mM KCl and 2 ml of buffer D containing 110 mM KCl, and the protein was then eluted with 3 ml of the same buffer containing 200 mM KCl. The sample was loaded on a 0.5-ml column of heparin-Sepharose equilibrated with buffer D containing 200 mM KCl. The column was washed with several quantities of this buffer, Fagomine manufacture and *AN/L3 was then eluted with sequential 1-ml portions of buffer D containing KCl in final concentrations of 0.25, 0.30, 0.35, 0.40, and 0.5 M. Proteins from each portion were analyzed by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE) followed by staining with Coomassie amazing blue and Western blot analysis. The fractions collected at 0.30, 0.35, and 0.4 M KCl were combined or dialyzed separately against buffer E (0.1 M KCl, 10 mM Tris-HCl [pH 7.5], 50% [vol/vol] glycerol, 1 mM DTT, 0.2 mM EDTA) and stored at ?20C for periods of 1 1 to 2 2 weeks or at ?80C for long-term storage. Protein concentrations were determined by SDS-PAGE followed by optical densitometry of the gel stained with Coomassie amazing blue. Bovine serum albumin (BSA) loaded in different Rabbit Polyclonal to Collagen V alpha2 amounts on separate lanes of the same gel was used for.