包含體純化步驟(精)-資料下載頁
2025-04-16 23:55本頁面
【正文】 g and discard supernatants.If larger culture volumes are required, refill microcentrifuge tube and centrifuge. Repeat this step until all cells are harvested.3. Resuspend cells in 200 μl buffer B. Lyse cells by gently vortexing, taking care to avoid frothing.The solution should bee translucent when lysis is plete. Most proteins are soluble in buffer B. If the solution does not bee translucent, lyse cells with buffer A.4. Centrifuge the lysate for 10 min at 15,000 g to remove the cellular debris, and transfer the supernatant to a fresh tube.5. Add 50 μl of a 50% slurry of NiNTA His?Bind Resin (25 μl resin has a capacity for 125250 μg His?Tag fusion protein) to each tube, and mix gently for 30 min at room temperature.6. Centrifuge 10 sec at 15,000 g to pellet the resin, transfer 10 μl of the supernatant to a fresh tube, and discard the remaining supernatant. Store the supernatant samples on ice.The supernatant samples will contain any proteins which have not bound to the resin.7. Wash the resin with 2 250 μl of buffer C.Centrifuge for 10 sec at 15,000 g between each wash step and carefully remove the supernatant.8. Elute the protein with 3 25 μl buffer E.Centrifuge for 10 sec at 15,000 g between each elution step and carefully remove the supernatant to a fresh tube.9. Analyze the fractions by SDSPAGE.If Buffer A was used to lyse the cells, refer to “Preparation of guanidine containing samples for SDSPAGE”, on page 27.
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