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Crystal shikonin (8 mg) was mixed with the cell lysate of MCF-7 (2 mg protein), rotated at 41C for 2 h and centrifuged to remove the supernatant. The precipitate (crystal
shikonin with bound cellular protein) was washed three times with chilled washing buffer (50 mMTris¨CHCl (pH 7.5), 150 mM NaCl, 2 mMEDTA, 10% glycerol, 1 mMNaF) by centrifugation (5000 r.p.m. for 1 min at 41C). The crystal shikonin with bound cellular protein was dissolved and boiled in SDS¨CPAGE sample buffer for 10 min. The samples were loaded and separated on 10% SDS¨Cpolyacrylamide gel and stained by silver staining. The bands were cut, digested in gel and loaded for LC-MS-MS spectrometer analysis by Shanghai Applied Protein Technology Co Ltd. Proteins were identified by searching the databases of Swiss-Prot using SEQUEST
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