User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/17

Asc Hb Project

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Purifying and Concentrating the Asc Hb with Nickel The protein was then continued to be purified over a PD-10 Desalting column: A PD-10 Desalting column was prepared with one column volume of dH2O was run over the column, and two column volumes of 25mM Tris, pH 8.3 run over the column. 2.5mL of the dialyzed Reconstituted Asc Hb was then run over the column. The protein was eluted off with 3.5mL of 25mM Tris, pH 8.3. The loading and elution of the protein was repeated many times. The protein was then run over a HiTrap Q FF column on an FPLC in the same way done with another Asc Hb with Ni(protoporphyrin IX) on 07/09/12. A Jumbosep™ Centrifugal Device was used to concentrate the protein: The fractions from yesterday's purification that had a significant absorbance at 280nm and 420nm were poured into the centrifugal device. The device was then spun at 3000g for 15 minutes at 4°C. The flowthrough was discarded (looked clear). More of these fractions were poured into the Jumbosep™ Centrifugal Device with the already concentrated protein. Again the device was then spun at 3000g for 30 minutes at 4°C. About 11mL of concentrated protein remained, which was saved and stored at 4°C. Concentrating the Asc Hb with Manganese A Jumbosep™ Centrifugal Device was used to concentrate the protein: The fractions from yesterday's purification that had a significant absorbance at 280nm and 420nm were poured into the centrifugal device. The device was then spun at 3000g for 30 minutes at 4°C. The flowthrough was discarded (looked clear). More of these fractions were poured into the Jumbosep™ Centrifugal Device with the already concentrated protein. Again the device was then spun at 3000g for 30 minutes at 4°C. About 8.5mL of concentrated protein remained, which was saved and stored at 4°C.