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.
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