06-06-2014, 04:01 PM
Workshop 7B- Protein Isolation and purification
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Introduction
The first step in protein purification involves a cell disruption step. The method of choice depends on the type of cell. In general, animal cells are easier to disrupt than bacteria, yeast or plant cells. The table below summarizes some of the methods. This list is by no means complete, as there are as many methods of disruption as there are types of cells
Once an appropriate method has been selected, the cells or tissues are suspended in an appropriate buffer with or without reducing agents, such as β-mercaptoethanol (20-30 mM) or
dithiothreitol (2-5 mM). After disruption is complete, the suspension is centrifuged at low speed (4,000-5,000 x g) to separate cell debris from the extracted and solubilized proteins. If a membrane preparation is desired, then the supernatant of the 4,000 x g centrifugation step is centrifuged at 30,000-100,000 x g, (the speed is dependent on the source of the cells). Membrane proteins can be extracted with various ionic or nonionic detergents (Deutscher).
Concentration and partial fractionation of a protein “extract”--The salting-out technique of protein purification is mainly dependent on the hydrophobic character of the protein. The salt is dissolved into the solution containing the protein. Water will solvate the added salt ions, decreasing the solvation of the protein itself. This decrease in solvation exposes the hydrophobic regions of the protein, which then interact with each other to form aggregates that will precipitate. In general, higher molecular weight proteins will precipitate out at lower salt concentrations. For this laboratory experiment, we will use ammonium sulfate. The optimum concentration of ammonium sulfate required to precipitate the protein of interest is determined by adding increasing amounts of the ammonium sulfate and saving the precipitate for further analysis. A table is included in this handout for preparing protein solutions of different concentrations of ammonium sulfate. A disadvantage of this method is the high amount of salt that must be removed from the precipitate. To remove the salt from the protein sample, we will use both dialysis and gel filtration chromatography
Sample: Bovine serum albumin and lysozyme in water
Procedure Ammonium sulfate precipitation
1. Pipet 3.0 mL of protein solution into a 50 mL centrifuge tube labeled 0-50 (be sure to put your name on the tube as well).
2. Weigh out the required amount of ammonium sulfate for 50% saturation at 0C (see table).
The initial concentration of ammmonium sulfate is 0%.
NOTE: The table uses grams/liter. Calculate the amount of ammonium sulfate needed for 3.0 mL of protein solution.
3. Add 1/3 of the ammonium sulfate to the 50 mL centrifuge tube, swirl the tube and allow to dissolve. Repeat with the remaining portions of ammonium sulfate.
4. Place the centrifuge tube on ice for 15 minutes.
5. Balance your tube with another student’s tube. Centrifuge for 10 minutes at 10,000 x gravity. Remember the position your tube was placed in the rotor.
6. Label a 15 mL tube “50-70”. Decant the supernatant from the 50 mL centrifuge tube into the
15 mL tube marked 50-70. Measure the volume of the 50-70 supernatant in the 15 mL tube and save for step 7. Dissolve the white precipitate (in centrifuge tube) in 1.0 mL of distilled water and transfer this to a microcentrifuge tube marked “Dialysis”.
7. Transfer the supernatant from the 15 mL tube labeled 50-70 into the 50 mL centrifuge tube.