Journal: The Journal of biological chemistry

Article Title: Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis *

doi: 10.1074/jbc.M311209200

Figure Lengend Snippet: A, SDS-PAGE and Western blot of the purified DesA3 enzyme. M, Bench-Mark™ prestained protein ladder (Invitrogen). Lane 1, DesA3 eluted from a 1 ml of metal BD-Talon™ resin chelating column (BD Biosciences) with 50 mM imidazole. Lane 2, DesA3 as eluted with 100 mM imidazole. Lane 3, pure DesA3 as eluted with 200 mM imidazole. Other details and staining of the gel are described under “Experimental Procedures.” B, DesA3-purified protein was added to the enzymatic reaction to determine the effect of purified DesA3 on Δ9 desaturation. A final volume of 1 ml of reaction mixture contained crude cell lysate of M. bovis BCG wild type (0.55 mg), 20 μl of a solution containing 50% Me2SO and 1% Tween 20, 1 μmol of NADPH in 0.1 mM potassium phosphate buffer, pH 7.2, and 25 nmol of [1-14C]18:0-CoA as substrate. No purified DesA3 was added to the assay (black bars), or purified DesA3 was added in the amount of 4.8 μg (gray bars). Duplicate assays were performed. After 1 or 2 h of incubation at 37 °C, the [1-14C]C18: 1-CoA was quantified as described in Fig. 6.

Article Snippet: Lane 1 , DesA3 eluted from a 1 ml of metal BD-Talon™ resin chelating column (BD Biosciences) with 50 mM imidazole.

Techniques: SDS Page, Western Blot, Purification, Staining, Incubation