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proteins
Selection of Tissue
Choice is based on the type of study Usually select tissue that has large amounts of the materials necessary for the study. Tissues are acquired in a fashion that provides for protection of the system to be studied.
Use fresh tissue or immediately placed in ice cold solution Bacterial cells are centrifuged at 16,000g and separated from growth media. The pellet(bacterial cells) is resuspended in buffer.
Gentle disruption
Osmotic shock: Mainly for cells without cell wall. (for cell wall digest with enzymes or snail gut juice before processing) Chemical solubilization: Cell membrane disrupted with detergents, such as triton x100, sodium cholate, or lysophospholipids or enzymes such as lysozymes, zymozymes , lipase Homogenizer: Mechanical disruption. A ground glass piston fits in ground glass cylinder. Piston moved up and down to rupture cells. Usually has water cooling system
Types of centrifuges
Analytical: used to determine the size of the molecule being studied Preparative: More common type. Has different type of motors
Fixed angle: most common with fixed angle. The sample tube is in a fixed angle. The force (x g) are reported for the average distance. Swinging bucket The buckets are able to swing up in to a position such that the sample goes directly down the length of the tube. This allows simple gradient. Samples are at the bottom of the tube rather than on the side.
200,000
10-24hrs
Membrane sheets
Precipitation of proteins
Salting out and IEP
Solubility in water
Proteins usually carry charge due to hydrophilic amino acids and terminal amine and acid groups. Because they have hydrophilic amino acids on their surfaces that attract water molecules and interact with them, proteins are soluble in water solutions.
Solubility in water
Solubility of proteins is a function of the ionic strength and pH of the solution. Proteins have isoelectric points at which the charges of their amino acid side groups balance each other.
Precipitation
The solubility is also a function of ionic strength
Salting-in: At low salt concentrations, the presence of salt stabilizes various charged groups on a protein molecule, thus attracting protein into the solution and enhancing the solubility of protein Salting-out: As you increase the ionic strength by adding salt, there is less and less water available for the protein to dissolve and proteins will precipitate.
Ammonium sulfate
Ammonium sulfate is the most common salt used:
Because it is unusually soluble in cold buffers (our extractions are kept cold!). In research laboratories as a first step in protein purification because it provides some crude purification of proteins separating non-proteins and some unwanted proteins out. Because it yields a precipitated protein slurry that is usually very stable, so the purification can be stopped for a few hours while the student gets some sleep
Other salts
Sodium sulfate Polyethylene glycol (PEG): organic polymer that has properties similar to ammonium sulfate
Change in pH
If the ionic strength is very low or very high, protein tend to precipitate at isoelectric point. In our case we add ammonium sulfate in two steps (reduce co-precipitation) and then crystallize by changing pH.