Protein Purification and Characterization.pptVIP

Protein Purification and Characterization Why Study proteins? IMPORTANT FACTORS IN PROTEIN PURIFICATION Starting materials tissues, cells or clones expressed in E. Coli or animal cells Decisions- quantity of protein, protein modification availability of samples, is it cloned yet, expense Stabilization of protein is key - proteins are not meant to be purified, so you need to keep them alive and happy (active / native) pH - both activity and structure are pH dependent Temperature - most stabile at low temperature - reduces energy in the system for unfolding and reduces the protease kinetics. Few proteins are unstable at low temps - ppdk (Dr. Chastains enzyme) and the ATPase in mitochondria Protease inhibitors - several classes of proteins catalyze the hydrolysis of peptide bonds (called proteases). Usually need to add several suicide inhibitors and reduce free metals which are used by the proteases Reducing agents - beta - mercaptoethanol and dithiolthreitol both act as reducing agents. Prevent the oxidation of amino acids Detergents - Membrane bound proteins often need added detergents (soaps) to mimic the ampipathic nature of the membrane you so cruelly ripped it from - need to be above the concentration at which micelles are formed - the critical micellular concentration (CMC) Homogenization - breaking the cell apart Mechanical Shearing - Warring blender, glass or plastic pestle like homogenizer Freeze Thaw - cycles under hypnotic conditions, ice crystals disrupts membranes METHODS OF PURIFICATION Centrifugation - Separation based on density, mass, shape and the density of the solution Sedimentation of particles measured by Svedburg units Force applied in gravitational (g) forces The centrifugal force depends on speed and time and radius of rotor Differential centrifugation One of the most used methods in biochemistry Uses increasing g forces to yield a pellet and a supernatant Subcellular centrifugation - a way to separate the cell contents based on density