After the reaction was stopped by adding 125 L of 20% perchloric acid, the reaction mixture was allowed to stand in an ice-cold water bath for 10?min, and centrifuged at 20,000for 5?min at 10?C
After the reaction was stopped by adding 125 L of 20% perchloric acid, the reaction mixture was allowed to stand in an ice-cold water bath for 10?min, and centrifuged at 20,000for 5?min at 10?C. results suggest that nephrosin promotes protection against bacterial infections7. Patristacin 10-Deacetylbaccatin III was first discovered in gulf pipefish (for 10?min to obtain supernatant fractions. The intestines from medaka was isolated and washed by pouring PBS into the gut. Intestine contents were thus collected in PBS. The gallbladder was isolated and perforated, and the bile was collected by draining. Western blot analysis was performed with the anti-OlPac2 or anti-OlPac3 antibodies. The proteins were transferred to a poly vinylidene di-fluoride (PVDF) membrane after separation by SDS-PAGE. The membrane was blocked with 2% BSA in TBST at 4?C overnight, followed by incubation with the primary antibody diluted in the blocking buffer (1:500 for OlPac2 and OlPac3) at room temperature for 2?h. After rinsing the membrane three times for 5?min with TBST, proteins were incubated with the secondary antibody, diluted 1:2000 in TBST, for 2?h. The membrane was rinsed five occasions for 5?min TBST, pre-equilibrated in 100?mM TrisCHCl pH 9.5 for 5?min, and developed with NBT/BCIP. Inclusion body refolding Inclusion bodies of OlPac2 and OlPac3 were dissolved in 50?mM TrisCHCl (pH 8.0), 8?M urea, 0.1?M 2-mercaptoethanol and 1?mM EDTA at 37?C for 20?min. After centrifugation at 20,000for 10?min, the supernatant was diluted in 8?M urea to a final protein concentration of 0.4?mg/mL. The protein answer was dialyzed against refolding buffer (50?mM TrisCHCl (pH 8.0), 0.8?M l(+)-arginine hydrochloride, 0.5?mM glutathione, 0.1?mM oxidized glutathione and 5?M ZnSO4) at 4?C for 2?days, and against 25?mM TrisCHCl (pH 8.0; once with 5?M ZnSO4 and twice without ZnSO4) at 4?C. The folding mixture was centrifuged at 20,000for 10?min, concentrated using Amicon Ultra 10?K filter models (Millipore, Billerica, MA), and the 10-Deacetylbaccatin III supernatant was used as the 10-Deacetylbaccatin III active enzyme. Partial purification of pactacin The intestines from 20 adult medaka were isolated, and the digestive 10-Deacetylbaccatin III fluid was washed by injecting 200 L of 10?mM TrisCHCl buffer (pH 7.5) twice into the gut. The digestive fluid thus collected was centrifuged at 15,000for 5?min, and the supernatant was filtered using a 0.45-m membrane. Next, one part of the fluid was directly applied to a Source 15Q (GE Healthcare Life Science, USA) column mounted in an HPLC system (Cilson, Middleton, WI, USA). The remaining fluid was incubated with 1?mM DFP at 30?C for 15?min prior to injection into the column. After the column was washed with a 10?mM TrisCHCl buffer (pH 7.5), the adsorbed protein was eluted with a Rabbit Polyclonal to TSC2 (phospho-Tyr1571) 10?mM TrisCHCl buffer (pH 7.5) employing a linear gradient (0C0.5?M) of NaCl concentration and a constant flow rate of 1 1?mL/min. Eluted fractions were collected for one minute each. Caseinolytic activity and protease inhibition Using 10 L of the eluted fractions, the caseinolytic activity was measured from their reaction with a buffered casein answer (375 L, 3.3?mg/mL casein, 83?mM TrisCHCl, pH 7.5)39. The mixture was incubated for 30?min at 30?C. After the reaction was stopped by adding 125 L of 20% perchloric acid, the reaction mixture was allowed to stand in an ice-cold water bath for 10?min, and centrifuged at 20,000for 5?min at 10?C. The absorbance 10-Deacetylbaccatin III at 280?nm of the supernatant was measured. For inhibition studies, eluted fractions (10 L) were incubated with an inhibitor (1?mM of HQ, OP, EDTA, DFP, or IAA) answer for 10?min at 30?C before testing caseinolytic activity as described above. Zymography Different fractions (peaks Ia, Ib, Ic, and II, and the synthesized rOlPac2) were mixed with SDS-PAGE buffer (2% SDS, 10% glycerol, and 0.01% bromophenol blue, 62.5?mM TrisCHCl pH6.8) without 2-mercaptoethanol, and incubated for 30?min at 80?C. Electrophoresis of the mixture was run on a 15% polyacrylamide gel made up of 0.1% casein. The gel was subsequently rinsed twice with 2% Triton X-100 in 20?mM TrisCHCl pH 7.5 buffer for 30?min, and once with 1% Triton X-100 in 50?mM TrisCHCl pH 8.0 buffer for 30?min. The gel was then incubated in 50?mM TrisCHCl pH 8.0 buffer containing 1?M ZnSO4 for 2?h at 30?C. Bands of casein degradation were visualized by staining the.