Extraction and Purification

1. Extraction and Purification proteins

2. 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.

3. Tissue and cell disruption • There are several method to disrupt cells and tissues depending on your tissue source, toughness, and what you wish to obtain • When the membrane is disrupted, two things are added • Buffer to main pH and compound dissolved in it to support cell osmotically • Antioxidant – dithiothreitol or β-mercatoethanol

4. Vigorous methods in total disruption of membranes • French press: A hydraulic pressure with special sample cell. Cells are pressured (20000 psi) and released through small orifice. This results in a shock wave that causes the cell to burst. Can choose right side out or inside out opening. This is the gentlest method. • Sonication: Uses the same principles as french press. Instead of a single shock wave there is a continuous flow of shock waves that causes the cells to burst repetitively. This results in mixed membrane vesicles. Often has higher yield than French press, and this system requires cooling samples. Microwaves are used and the samples are pulsed and cooled several times.

5. Vigorous methods in total disruption of membranes • Polytron, tissumizer: Consists of rotating blade within a shaft that can be forced into the cell suspension or tissue suspension. The technique uses shear forces to disrupt cells • Bead Mill, shaker: cells are broken in a container with beads. The container is shaken vigorously. The system can generate considerable heat and so usually has cooling coils around it.

6. Moderate method for cell or tissue disruption • Blender: This is classical waring blender. Used mostly for plant and connective animal tissues. • Grinding with abrasive: Cells are placed with sand or some other abrasive material and ground in mortar and pestle. Good method for tough cells like yeast but is tedious

7. 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

8. Separation of large parts • Once cells are disrupted, either we have to collect parts we need or remove unwanted parts. • FILTRATION • Cheese cloth: mainly removes materials that are not homogenized • Screens: calibrated wire screens can separate broken and non-broken eukaryotic cells • Filters: Variety of filters are available from 0.22µm to some that will allow proteins molecules less than 3000 to pass are available. Problem – plugging can be reduced by tangential flow.

9. Separation of large particles • Centrifugation: Accelerated sedimentation. Sedimentation rate depends on several factors. • Force pushing down due to gravity F= mg • Opposing forces • Buoyancy density of the particle and the buoyant force or the density of the solvent • Frictional force depends on shape of the particles and the material with which it is going.

10. 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. Types of centrifuges

11. Separation of cellular components • Differential centrifugation:

12. Separation of cellular components • Density gradient centrifugation; • Exploits the different density of organelles • Density gradients are formed by using sucrose as solute • Can be step gradient or continuous • Centrifuge for set time at a know force and determine where your compound is or run it until it reaches equilibrium. Sample will stop moving once its density is equal to solvent’s density.

13. Precipitation of proteins Salting out and IEP

14. 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.

15. 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.

16. 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.

17. 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

18. Other salts • Sodium sulfate • Polyethylene glycol (PEG): organic polymer that has properties similar to ammonium sulfate

19. 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.