Leishmania Cm contains enriched proteins and is minimally contaminated by incidental cell lysis. (a) Leishmania promastigotes were metabolically labeled, as described in Materials and methods. Conditioned medium (Cm) from labeled cells and the cells themselves were collected in parallel, and the proteins present in the Cm and corresponding whole cell lysate (WCL) of promastigotes were precipitated in 10% trichloroacetic acid (TCA). Equal numbers of TCA-precipitated counts/minute of Cm and WCL were fractionated on a 5% to 20% gradient polyacrylamide gel. Arrows indicate proteins specifically enriched in leishmania Cm. The autoradiograph shown is representative of three independent experiments. (HMW) High molecular weight marker. (b) To control for inadvertent lysis of organisms during collection of Cm, glucose-6-phosphate dehydrogenase (G6PD) activity in Cm collected from isotopically labeled and nonlabeled cells was measured as described in Materials and methods and compared with the activity associated with deliberately lysed promastigotes. The data shown are the means of measurements from three independent experiments. 0.01 units of G6PD were assayed as a control in each experiment. The asterisks shown indicate a significance difference when compared with Cm (P < 0.001), calculated by one-way analysi of variance followed by Bonferroni's correction for multiple comparisons (GraphPad Prism 4.0).
Silverman et al. Genome Biology 2008 9:R35 doi:10.1186/gb-2008-9-2-r35