User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/17: Difference between revisions
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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 dH<sub>2</sub>O 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 [[User:Tamra L. Fisher/Notebook/Research for Dr. Hartings/2012/07/09| 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 8.5mL 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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Revision as of 12:21, 17 July 2012
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Purifying and Concentrating the Asc Hb with Nickel
Concentrating the Asc Hb with ManganeseA Jumbosep™ Centrifugal Device was used to concentrate the protein:
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