User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/02: Difference between revisions
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** Imidizole also contains the functional group of histidine. | ** Imidizole also contains the functional group of histidine. | ||
* We needed to first run the binding buffer, followed by the elution buffer, and then the binding buffer once more through the column otherwise the column will have too much imidazole. | * We needed to first run the binding buffer, followed by the elution buffer, and then the binding buffer once more through the column otherwise the column will have too much imidazole. | ||
* The column only run at 5mL/minute. The nickel column of the instrument can hold 5mL and contains polymer beads. The ADA protein contains specifically a histag on the protein of 6 histidines. Histidine has an affinity to nickel and therefore will bind with it. | * The column only run at 5mL/minute. The nickel column of the instrument can hold 5mL and contains polymer beads. The ADA protein contains specifically a histag on the protein of 6 histidines (which is the amount necessary in order to have a night enough affinity to nickel). Histidine has an affinity to nickel and therefore will bind with it. | ||
* We then put the protein on. We ran sample 1 first. | |||
* The protein histidines (in the ADA) bind with the nickel in the column. To get the protein out, we use the elution buffer because the imidazole histidines compete with the histidine in the column and binds to the nickel. This results in the protein being pushed out. | |||
Revision as of 00:13, 3 October 2012
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FPLCGoalsRun an FPLC of the protein in order to purify it. Procedure
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