tailieunhanh - Ebook Principles and techniques of biochemistry and molecular biology (7th edition: Part 2

(BQ) Part 2 book "Principles and techniques of biochemistry and molecular biology" presents the following contents: Protein structure, purification, characterisation and function analysis, mass spectrometric techniques, electrophoretic techniques, chromatographic techniques, spectroscopic techniques - II spectrophotometric techniques,. Invite you to consult. | 337 Protein structure determination attempted using as homogeneous a preparation as possible such preparations having a greater chance of yielding crystals than material that contains impurities. Because of our inadequate understanding of the physical processes involved in crystallisation methods for growing protein crystals are generally empirical but basically all involve varying the physical parameters that affect solubility of the protein-for example pH ionic strength temperature presence of precipitating agents-to produce a state of supersaturation. The process involves extensive trial and error to find a procedure that results in crystals for a particular protein. Initially this involves a systematic screen of methods to identify those conditions that indicate crystallinity followed by subsequent experiments that involve fine-tuning of these conditions. Basically nucleation sites of crystal growth are formed by chance collisions of molecules forming molecular aggregates and the probability that these aggregates will occur will be greater in a saturated solution. Clearly to produce saturated solutions tens of milligrams of proteins are required. This used to represent a considerable challenge for other than the most abundant proteins but nowadays genetic engineering methodology allows the overproduction of most proteins from cloned genes almost on demand. The following are some of the methods that have proved successful. a Dialysis. A state of supersaturation is achieved by dialysis of the protein solution against a solution containing a precipitant or by a gradual change in pH or ionic strength. Because of frequent limitations on the amount of protein available this approach often uses small volumes 50 mm3 for which a number of microdialysis techniques exist. b Vapour diffusion. This process relies on controlled equilibration through the vapour phase to produce supersaturation in the sample. For example in the hangingdrop method a microdroplet 2-20 mm3 of