User:Mary Mendoza/Notebook/CHEM 571 Experimental Biological Chemistry I/2012/09/25

Lysis of transformed E. coli

• To retrieve the adenosine deaminase (ADA) from E.coli, the cell must be lysed. By lysing the cell, the cytoplasm will no longer isolate the macromolecules, such as the ADA for this instance, from the extracellular environment.
• Samples 1 and sample 2 which were placed below -80°C on 9/19/12 is taken out from the freezer to thaw to room temperature.
• There are different methods by which the cell can be lysed. These methods include heat shock, blender polytron, homogenization etc. For this procedure, sonication will be used to lyse the cell. By exposing the cells to high frequency sound waves of a sonicator, the cell membrane are sheared exposing the intracellular contents.
• The sonicator used is the Misonix XL-2000 series Ultrasonic Liquid Processors.
• When the samples were at room temperature, a bucket of ice was prepared.
• Sample 1 was uncapped and inserted through the nozzle of the sonicator for 30 sec. followed by a 30 sec. soak in an ice bath. The nozzle is wiped off with kim wipes. Cooling the sample in an ice bath is necessary to prevent the denaturing of proteins. Denaturation can occur due to the heat generated by the high frequency sound waves of the sonicator.
• While sample 1 is in the ice bath, sample 2 was inserted through the nozzle. These steps were repeated two more times with a total repetition of 3 for each sample.
• Upon finishing sonication of cells, the samples were transferred to centrifuge tubes. The rotor chosen for the tubes is the make of Fiberlite (Piramoon Technologies Inc.) rotor F21-8x50y max. 21,000 rpm.
• The samples contained in the centrifuge tubes were balanced on the rotor having one of the tubes contain water (total of four tubes). The centrifuge settings were fixed on 18,000 rpm at 4°C for 2 h.
• The centrifuge is necessary to separate the water soluble from the water insoluble contents. The supernatant is the desired phase from this centrifuge because this contains the water soluble proteins (ADA) expressed by E. coli.

Filtration of Buffers

Assembly of Membrane Filtration Image Source: http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/counting.html

• The assembly shown in the picture on the right was followed with an additional clip that would hold the filter tightly to the flask. The sidearm of the 1000 mL flask was connected to a Welch vacuum pump.
• The membrane filter had a diameter of 47mm with a 450 nm pore diameter.
• The pump was turned on; the knob was unscrewed. The setting was set to 60 cmHg vacuum. This setting was chosen at random.
• The first pour was done with the binding buffer. This was done to ensure that the buffer with the lower imidazole content (binding) would not be contaminated with the buffer that had the higher imidazole content (elution).
• The collected filtrate of the binding buffer was poured into a sterilized glass bottle. This was followed by the pouring of the elution buffer into the filtering system. The filtrate for the elution buffer was also poured into a sterilized glass bottle.
• The bottles were capped with aluminum foil tops and refrigerated. The buffers were clear, colorless liquid solution before and after filtration.
• The filtrates were poured into their respective falcon tubes and refrigerated.

Filtration of the Supernatant

• The centrifuge tubes were taken out from the chamber. The supernatant of the tubes were poured into the filtering system. Sample 1 remained isolated from sample 2 which contained pellets 2, 3, and 4 from last week.
• The current pellet was left untouched in the centrifuge tubes. This are discarded material that contains disrupted cell organelles and other cellular material.