Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/22 July 2015"
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'''<u>Start of IMAC Purification</u>''' | '''<u>Start of IMAC Purification</u>''' | ||
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| + | 4mL of lysate was added to the resin saved from last time's run through of batch purification. However, due to a miscommunication, the resin from last time was suspended in strip buffer instead of 1X binding buffer. The resin was not re-charged yet with nickel and therefore unable to function properly. The lysate, diluted in 1X binding buffer, was added to this ill-prepared resin, and the supernatant collected after the first centrifugation, fraction 1, appeared extremely green. The resin was white instead of the usual blue, indicating that the resin beads were not charged and therefore unable to bind Histidine-tagged proteins like our Tamura. The supernatant was still collected into a tube. The resin was washed thoroughly with about 10mL of DI water, spun for 2 minutes at 700g and 4 degrees Celsius. The supernatant was collected and discarded. Next, the resin was charged with about 10mL of charge buffer, fully incorporated by spinning on a rotary spinner, and then centrifuged for 2 minutes at 700g and 4 degrees Celsius. The supernatant was once again discarded. Finally, 10mL of 1X binding buffer was added to the resin, and the same process of incorporation and centrifugation was carried out. | ||
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| + | The lysate, diluted in 1X binding buffer, was re-added to the freshly prepared resin, and batch purification proceeded as usual. However, after the first centrifugation step, the resin appeared white and the supernatant appeared blue. This qualitative observation was unprecedented. The resin was no longer charged and the nickel beads had somehow spun off and became incorporated into the supernatant instead. The Falcon tube's contents were vortex-ed and re-spun on a rotary spinner. We tried centrifugation two more times, but observed the same result. We conclude that the contents of the resin must have been somehow irreversibly disrupted when we added our lysate to the ill-prepared resin. | ||
| + | The resin seemed unsalvageable. Tomorrow, 2mL of fresh resin will be used and IMAC purification will be attempted again. | ||
Latest revision as of 20:16, 24 July 2015
4mL of lysate was added to the resin saved from last time's run through of batch purification. However, due to a miscommunication, the resin from last time was suspended in strip buffer instead of 1X binding buffer. The resin was not re-charged yet with nickel and therefore unable to function properly. The lysate, diluted in 1X binding buffer, was added to this ill-prepared resin, and the supernatant collected after the first centrifugation, fraction 1, appeared extremely green. The resin was white instead of the usual blue, indicating that the resin beads were not charged and therefore unable to bind Histidine-tagged proteins like our Tamura. The supernatant was still collected into a tube. The resin was washed thoroughly with about 10mL of DI water, spun for 2 minutes at 700g and 4 degrees Celsius. The supernatant was collected and discarded. Next, the resin was charged with about 10mL of charge buffer, fully incorporated by spinning on a rotary spinner, and then centrifuged for 2 minutes at 700g and 4 degrees Celsius. The supernatant was once again discarded. Finally, 10mL of 1X binding buffer was added to the resin, and the same process of incorporation and centrifugation was carried out.
The lysate, diluted in 1X binding buffer, was re-added to the freshly prepared resin, and batch purification proceeded as usual. However, after the first centrifugation step, the resin appeared white and the supernatant appeared blue. This qualitative observation was unprecedented. The resin was no longer charged and the nickel beads had somehow spun off and became incorporated into the supernatant instead. The Falcon tube's contents were vortex-ed and re-spun on a rotary spinner. We tried centrifugation two more times, but observed the same result. We conclude that the contents of the resin must have been somehow irreversibly disrupted when we added our lysate to the ill-prepared resin. The resin seemed unsalvageable. Tomorrow, 2mL of fresh resin will be used and IMAC purification will be attempted again.