![]() ![]() ![]() Vortex briefly to wash the beads and pool this wash with the first cell lysate.Ĭentrifuge for 10 min, 16,000 x g at 4oC to pellet the precipitated proteins and cell debris. Optional: Add 100 uL of fresh TCA buffer to the original microfuge tube that contains the glass beads. Transfer the cell lysate to a new microfuge tube on ice. If the cell pellet is quite large, using too small a volume at this step will reduce the overall yield of protein. ![]() As proteins will be pelleted later on, larger volumes of TCA buffer and beads can be used to break open cells if desired. The resuspension and wash volumes can be varied depending on your preference. Chill tubes on ice for 3 mins between vortexing to keep cells chilled. When thawed add half volume of glass beads and vortex using a multi-vortexer in 5 x 1 min bursts. To frozen pellet add 300 uL of TCA buffer, on ice. Where this is so, the final volume of resuspension buffer used to resuspend the protein pellet should be scaled down accordingly. Typically 10 mL of early-log phase (OD600nm 0.6-1.0) culture should be used for the starting material, but smaller culture volumes can be used. Freeze cell pellet in liquid nitrogen and store at -70oC. Because of the very low pH of the TCA solution, the proteins are not readily degraded by proteases during the purification procedure.Ĭollect cell pellet for extraction by centrifugation at 4oC. Yeast proteins can be isolated efficiently and directly from intact cells by acid extraction using a 10% TCA solution. ![]()
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