User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06: Difference between revisions
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Temperature: 4°C | Temperature: 4°C | ||
Time elapsed: 2 hours | Time elapsed: 2 hours | ||
Rotor used: | Rotor used:_____ | ||
* After centrifugation, supernatants and pellet form. The pellet was discarded, and about 100mL supernatant yielded and was kept in a 500mL beaker. | * After centrifugation, supernatants and pellet form. The pellet was discarded, and about 100mL supernatant yielded and was kept in a 500mL beaker. | ||
* The supernatant was filtered through Supor®-450 47mm membrane filter by pouring the supernatant through filter with membrane attached. Filter was attached to pressure suction that draws the supernatant through the membrane filter. The supernatant was filtered into a 800mL Erlenmeyer flask, then poured into a 25mL falcon tube. | * The supernatant was filtered through Supor®-450 47mm membrane filter by pouring the supernatant through filter with membrane attached. Filter was attached to pressure suction that draws the supernatant through the membrane filter. The supernatant was filtered into a 800mL Erlenmeyer flask, then poured into a 25mL falcon tube. | ||
* The filtered protein supernatant was injected into the GE Pharmacia® AKTA Purifier 100 Fast Protein Liquid Chromatography to separate ADA proteins from the rest of proteins in sample through the following procedure: | * The filtered protein supernatant was injected into the GE Pharmacia® AKTA Purifier 100 Fast Protein Liquid Chromatography to separate ADA proteins from the rest of proteins in sample through the following procedure: | ||
** | ** The column was ran using elution buffer with _____ at a speed of ___mL/s | ||
** Protein samples were injected into FPLC with a 10mL syringe and let run through the column at a speed of _____ | |||
** Binding buffer was run through the column with _____at a speed of_____ | |||
** UV-vis spectrum was taken at the eluted product over time and chromatogram was yielded. Because the chromatograph was not saved, the data cannot be displayed here. | |||
** Fractions of protein was collected based on the UV-vis spectrum, and three separate fractions are formed as a result of affinity binding and competitive binding via imidazole. | |||
** Fraction 1 and 3 from the column were collected and transferred into a 15mL falcon tube | |||
** Fraction 2 from the column, which were suspected to obtain the highest concentration of ADA protein, were transferred into a separate 15mL falcon tube. | |||
==Result for Protein Extraction and Purification from BL21(DE3) Cells== | ==Result for Protein Extraction and Purification from BL21(DE3) Cells== |
Revision as of 20:30, 24 November 2012
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Purpose
Procedure for Protein Extraction and Purification from BL21(DE3) Cells
Speed: 18,000rpm Temperature: 4°C Time elapsed: 2 hours Rotor used:_____
Result for Protein Extraction and Purification from BL21(DE3) Cells
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