Chapter 3 - Chapter 3 3.6 Protein Purification Techniques...

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Chapter 3 3.6 Protein Purification Techniques Purifications steps usually exploit minor differences in the solubilities, net charges, sizes, and bninding specificities of proteins. Most purification techniques are performed at 0 C to 4 C to minimize temperature dependent processes such as protein degradation and denaturation. First step in protein purification is to prepare a solution of proteins. Isolation of an intracellular protein requires that cells be suspended in a buffer solution and homogenized, or disrupted into cell fragments. Under these conditions, most proteins dissolve. Next step in protein purification is often a relatively crude separation, or fractionation, procedure that makes use of the different solubilities of proteins in salt solutions. Target proteins and other, more soluble proteins remain in the fluid, called the supernatant fraction. Mixture is centrifuged, the fluid removed, and the precipitate dissolved in a minimal volume of buffer solution. Ammonium sulfate gives a two- to- threefold purification. Solvent containing residual ammonium sulfate is exchanged by dialysis for a buffer solution suitable for chromatography. Cylinder of cellophane tubing. Column choromatography can then be used to fractionate the mixture of proteins that remains after ammonium sulfate precipitation and dialysis. As solvent flows through the column, the eluate (the liquid emerging from the bottom of the column) is collected in many fractions. Depends on interactions between matrix and protein. Different proteins are eluted at different rates. The concentration of protein in each fraction can be determined by measuring the absorbance of the eluate at a wavelength of 280 nm.
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Column chromatography may be performed under high pressure using small, tightly packed columns, with solvent flow controlled by a computer. HPLC Ion-exchange chromatography, the matrix carries positive charges or negative charges. Bound proteins can be serially eluted by gradually increasing the salt concentration in the solvent. Reaches a concentration where the salt ions out compete proteins in binding to the matrix. Protein is released and is collected in the eluate. Individual bound proteins are eluted at different salt concentration, and
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