PMC:2728203 / 24619-25683 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2728203","sourcedb":"PMC","sourceid":"2728203","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2728203","text":"His-CT-Mlp1 (pAC1486) was expressed in E. coli DE3 cells. Cells were collected and lysed in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, and 10 mM imidazole, pH 7.0) supplemented with protease inhibitor mixture by incubation with lysozyme and sonication. Lysates were clarified by centrifugation and incubated with Ni–NTA agarose (Qiagen) in lysis buffer for 2 h at 4 °C with mixing. The beads were then washed with wash buffer (50 mM NaH2PO4, 300 mM NaCl, and 20 mM imidazole). His-Nab2 and His-CT-Mlp1 were eluted from agarose with 250 mM imidazole. Sepharose-bound GST or GST-CT-Mlp1 (6 μg) was incubated with 2 μg of purified His-Nab2 at 4 °C in PBS for 90 min. Sepharose-bound GST-NT-Nab2 WT, F72D, or F73D (6 μg) was incubated with purified His-CT-Mlp1 (2 μg). Unbound fractions were collected and the beads were washed three times with PBS. Bound fractions were eluted with sample buffer (50 mM Tris–HCl, pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA, and 0.02% bromophenol blue) and analyzed by SDS-PAGE followed by Coomassie Blue staining.","tracks":[]}