3590). The resulting absorbance was measured at 595 nm in a Bio-Tek EL808 plate reader. The presence of compounds that competed with CAS for

metal binding caused a reduction in absorbance. SIS3 ic50 Changes (reductions) Bortezomib in absorbance were measured relative to the most strongly absorbing fraction in the column profile and plotted as indicated. Preparative TLC procedures Preparative TLC separations were performed on Avicel® Microcrystalline Cellulose Plates (20 × 20 cm, 1000 μm layer). Prior to use, preparative plates were washed by ascending chromatography in deionized water (twice) followed by one wash with redistilled 95% ethanol. The plates were dried overnight between washings. The chromatographic samples consisted of 2.0-mL aliquots per plate of a concentrated 76% ethanol solution (40× concentration) of the solids from the main Cu-binding peak of the Sephadex G-15 fractionation described in the text. The Analtech TLC Sample Streaker was used to apply the sample

by repeated streaking across an origin line located 3 cm buy PXD101 from the end of the plate. A filtered air-stream was used to dry the origin for 20 to 30 seconds between applications. Following the last application and prior to development, the plates were allowed to air dry for 8 minutes outside the stream. The plates were developed in 76% (v/v) ethanol (250 mL solvent in rectangular tanks, dimensions ca. 30L × 10W × 26 cm H) over a distance of 12 cm, dried, and examined under UV light (254 nm). Preliminary

experiments determined that the ninhydrin-reactive compound of interest was localized in a narrow band (ca. 1 cm diameter, ca. Rf 0.55) delineated at its leading margin by a narrow UV-absorbing band and bounded at its trailing edge by a narrow fluorescent band immediately Thymidine kinase preceding a broader UV-absorbing band. These bands were used as markers in purifying the compound by preparative chromatography. Eight preparative thin-layer plates were used to fractionate the 40× solution of the material recovered from Sephadex G15 column. The plates were developed with 76% ethanol. For each chromatogram, the area between the UV-absorbing and UV-fluorescent marker bands was scraped into separate 30-mL Corex centrifuge tubes. Deionized water (10 mL per tube) was added to each tube. After the tubes were vortexed (3 min), an additional 10 mL of deionized water was added to each tube, and the tubes were centrifuged at 6800 × g in a Sorvall SS34 rotor. The supernatants were decanted, pooled, filter sterilized [0. 2-μm, 25-mm Acrodisc syringe filter (Pall Life Sciences, Ann Arbor, MI)], and stored at 4°C prior to final purification by Sephadex G-15 column chromatography. Structural analysis of purified compound NMR data were acquired on a Bruker DRX 300 MHz spectrometer equipped with a 5 mm BBO NMR probe.