User:Puja Mody/Notebook/Chem 571: Gold Nanoparticles/2012/10/02: Difference between revisions
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==Procedure== | ==Procedure== | ||
*FPLC is Fast protein liquid chromatography and is used in order to purify the protein and uses a system of pumps in order to push the various liquids around the apparatus. | *FPLC is Fast protein liquid chromatography and is used in order to purify the protein and uses a system of pumps in order to push the various liquids around the apparatus. | ||
*First, the system needed to be equilibrated. The superloop pushed proteins through the system and can hold 50 mL. In A<sub>1</sub> the tube was placed in the binding buffer and for B<sub>1</sub> the tube was placed in the elution buffer in order to equilibrate the system. | |||
* We needed to run 25mL of Buffer through the column at a pressure of .14/.15 ( the pressure tells if there is anything clogging the system) | |||
** The conductivity meter reads how much salt is in the system | |||
** 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. | |||
* 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. | |||
Revision as of 00:10, 3 October 2012
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FPLCGoalsRun an FPLC of the protein in order to purify it. Procedure
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