Figure 1From: Cytotoxic activity of proteins isolated from extracts of Corydalis cava tubers in human cervical carcinoma HeLa cells Protein purification and electrophoretic analysis. (A) Chromatographic profile of protein purification from C. cava tuber extracts in HT Heparin column (GE Healthcare). Fractions 1 to 9 represented flow-through fractions, 10 to 27 were eluted with a linear NaCl gradient (from 0 to 2 M). The absorbance of all fractions was measured at 280 nm and their DNase activity was estimated using in-gel assay. Proteins present in fractions 16, 17 and 18 were identified using LC-ESI-MS/MS. Fractions 16-19 following three rounds of purification were used in tests on HeLa tumour cell line. (B) In-gel DNase pattern of the gel in which DNase activity of protein fractions originating from C. cava tuber extracts was estimated. ssDNA containing 10% SDS-PAGE, following electrophoresis and incubation (12 h) in 10 mM Ca2+buffer, pH 8.0, was stained with ethidium bromide and viewed under UV light. Nucleolytic activity was noted in fractions Nos.16, 17 and 18 of MW around 30 kDa. Control: purified nucleases from Chelidonium majus milky sap served as positive control. (C) 10% SDS-PAGE following electrophoresis of fraction-contained proteins and silver staining according to Shevchenko et al. [20]. The separated fractions were identical to those in the gel in Fig. 1B. Fractions nos. 16 and 17 each contained 5 protein bands of MW around 30, 32, 35, 38 and 68 kDa, while fraction no. 18 contained an additional band of MW around 140 kDa. The protein bands were numbered 1-6, excised from the gel and sent for identification by mass spectrometry (LC-ESI-MS/MS). M - Protein Molecular Weight Marker (Fermentas).Back to article page