Purification of WW domain

All the processes except the final reverse chromatography were carried out at 4°C. After the growth was saturated, the total LB medium was centrifuged to collect the cells. The cells were disrupted by sonifier (Branson) and again centrifuged to precipitate the needless cell debris. The supernatant was loaded onto glutathion sepharose (Amersham Bioscience) and washed and eluted as written in manufacturer's guide. The PreScission protease (Amersham Bioscience) was added into the eluted GST-WW domain and the solution was incubated at 4°C until the digestion was completed. The digested GST and WW domain was loaded to the Resource-RPC column (Amersham Bioscience) equilibrated by the solution composed of 0.1% TFA and 1% acetonitrile. The elution was carried out with gradual increase of acetonitrile concentration. The purified WW domain of CA150/FBP28 was analyzed by MALDI-TOF-MS on a Voyager mass spectrometer (PE) and the purity was also checked with SDS-PAGE.

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