User:Keyun Wang/Notebook/Experimental Biological Chemistry I/2012/11/06

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Purpose

  • Protein was extracted from transfected BL21(DE3) E.coli cells and adenosine deaminase was purified via column chromatography.
  • Adenosine deaminase plasmid with the mutation E34K was transformed into BL21(DE3) E.coli cells with a second attempt.
  • UV-vis spectroscopy was used to analyze Au/Lysozyme solutions made on 2012/10/31 to measure absorbance of supernatant of Au/Lysozyme samples

Procedure for Protein Extraction and Purification from BL21(DE3) Cells

  • Frozen BL21(DE3) E.coli cells from 2012/11/04 were taken out from the freezer with gloves.
  • The frozen cells were placed into a 500mL beaker filled with ~30°C of warm tap water for 10 minutes. After 10 minutes, cells are completely thawed and appeared in liquid form.
  • On ice bath, the cells were sonicated in three 1 minute intervals with three repeated cycles of procedures below:
    • Cells are sonicated under 8mV for 30 seconds with ______Sonicator.
    • Cells are immediately placed on ice for 30 seconds
  • After sonication, the E.coli cells were transferred from a 25mL falcon tube into a 30mL centrifuge tube. Another 30mL centrifuge tube was filled with tap water with the mass equal to the mass of 30mL centrifuge tube containing cellular sample.
  • The sample and water weight for balance were centrifuged in _______instrument with the following conditions:
    Speed: 18,000rpm
    Temperature: 4°C
    Time elapsed: 2 hours
    Rotor used:
  • 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 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:

Result for Protein Extraction and Purification from BL21(DE3) Cells

Au/Lysozyme ratio Observation of solution
20 pale and transparent purple, no fibers are formed
30 pale and transparent purple, no fibers are formed
40 pale and transparent purple, no fibers are formed
50 transparent purple in color, no fibers are formed
60 medium transparent purple in color, no fibers are formed
70 Clear supernatant, purple fibers formed aggregating at the bottom of test tube
80 Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant.
100 Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant.
120 Clear supernatant, purple fibers formed aggregating at the bottom of test tube. Some fibers were floating in supernatant.
130 Clear supernatant, purple fibers formed aggregating at the bottom of test tube



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