Leishmania HSPs are enriched in Cm. Leishmania conditioned medium (Cm) was collected from a culture containing about 2 × 109 promastigotes. The proteins contained therein were precipitated and then solubilized directly in Laemmli sample buffer. One half of this volume (containing a known amount of protein) was loaded into the lane labeled 'Cm'. From the 2 × 109 promastigotes recovered from the culture, 5% (1 × 108) were removed and processed in parallel with the remaining 95%. The 5% figure was chosen based upon the estimated (see Figure 1) maximum number of organisms that may have undergone incidental lysis during the incubation period and the collection centrifugation. Proteins contained in the whole cell lysate (WCL) prepared from the 1 × 108 cells were precipitated and resolubilized in a volume equal to that used to resolubilize the proteins precipitated from Cm collection. To allow for direct comparison to be made, half of this volume was then loaded into WCL lane #1. Protein was also precipitated from the WCL prepared from the remaining 95% of the cells and after solubilization in sample buffer an amount of protein was loaded into WCL lane #2 equal to that loaded into the lane labeled Cm. After transfer to nitrocellulose membrane, blots were probed, stripped, and reprobed with the indicated antibodies. The data shown are representative of results obtained in at least three identical experiments. EF1alpha, elongation factor-1α; Hsp, heat shock protein; SAcP, secreted acid phosphatase.