Journal: The Journal of biological chemistry

Article Title: Lipid dependence and basic kinetics of the purified 1,2-diacylglycerol 3-glucosyltransferase from membranes of Acholeplasma laidlawii.

doi: 10.1074/jbc.272.2.929

Figure Lengend Snippet: FIG. 1. Purification of the 1,2-diacylglycerol 3-glucosyltrans- ferase in three column chromatography steps. Protein (E) and MGlcDAG synthesis activity (G) were eluted in three different column chromatography steps as described under “Materials and Methods.” A, IEC on SP-Sepharose (Pharmacia), pH 8. The solid black line is the applied NaCl concentration. B, gel filtration on Sephacryl S-100 HR (Pharmacia); one of the four best fractions from the former IEC step. C, HAC on ceramic hydroxyapatite Macro-Prep (Bio-Rad), pH 8. The solid black line is the applied KH2PO4/K2HPO4 concentration. The MGlcDAG synthesis activity was assayed as described under “Materials and Meth- ods” with 27 mM amphiphiles present (3.7 mol % DOG, 33 mol % DOPG, and 63 mol % CHAPS).

Article Snippet: Hydroxyapatite Chromatography (HAC)—A column (16 mm in diameter) packed with 6 ml of ceramic hydroxyapatite Macro-Prep (Bio-Rad) was equilibrated with HAC-buffer: 100 mM KH2PO4/K2HPO4, pH 8, 20% (v/v) glycerol, and 20 mM CHAPS.

Techniques: Purification, Column Chromatography, Activity Assay, Concentration Assay, Filtration

Journal: Journal of Biological Chemistry

Article Title: Translocation of Group 1 Capsular Polysaccharide in Escherichia coli Serotype K30

doi: 10.1074/jbc.m308775200

Figure Lengend Snippet: FIG. 3. Purification of Wza and WzaHis6. The purification protocols are described under “Experimental Procedures,” and the figure shows a Coomassie Blue-stained SDS-PAGE gel of relevant fractions. Lanes 1–4 show the purification of Wza from SB 3–14-solubilized OM (lane 1) by sequential anion exchange chromatography (lane 2) and hydroxyapatite chromatography (lane 3). All three lanes show Wza migrating as a major band at 40 kDa (i.e. Wza monomer) when the samples are heated in SDS-sample buffer at 100 °C prior to electrophoresis. The purified Wza sample from lane 3 shows the multimeric form when the samples are run in the same buffer without heating (lane 4). Lanes 5 and 6 show heated and unheated samples of purified WzaHis6, respectively, confirm- ing that WzaHis6 also forms SDS-stable multimers.

Article Snippet: Fractions containing Wza were applied to a hydroxyapatite column (5-ml Econo-PacCHT-II, Bio-Rad) equilibrated with 50 mM sodium phosphate buffer, pH 7.0, containing 0.008% DDM and the protein was eluted with a linear gradient of 0.05–1 M sodium phosphate buffer, pH 7.0, containing 0.008% DDM.

Techniques: Purification, Staining, SDS Page, Chromatography, Electrophoresis