MCB+102+F10 lecture 04

MCB+102+F10 lecture 04 - I. Natural source Cells must be...

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Unformatted text preview: I. Natural source Cells must be ruptured and the proteins released by various mechanical means: Sonication French or Eaton pressure cell Waring blender Grinding with abrasive agent (alumina, micro-glass beads) The Concept of an Appropriate Starting Material II. Recombinant" source Amplification of desired protein by use of the cloned gene in an appropriate expression vector The Concept of a Protein Purification Buffer An aqueous mixture of components that extracts the desired protein into solution and helps preserve the structure and function of the desired protein because it resists any change in pH (it contains equimolar amounts of a conjugate acid-base pair that has a pK a value at or very near physiological pH) and because it contains other reagents that help prevent the desired protein from suffering unwanted oxidative and/or proteolytic damage. Typical Buffer Components: Buffering species (to hold pH constant) Salt (proteins are more soluble at moderate ionic strength) Reducing agent (to prevent oxidation of Cys) Chelator (to remove heavy metal ions) Protease inhibitors (to prevent proteolysis) Other (divalent cation, cofactor, stabilizer) 11 10 9 8 6 5 7 (of 0.1 mM HCl) : H + Cl- Triethanolamine (TEOLA) TEOLA-HCl pK a 7.6 Pure Water 100 mM TEOLA buffer pH is most resistant to change when it is equal to the pK a of the buffering species Use of a Buffering Species to Maintain Constant pH The "salting in" effect...
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MCB+102+F10 lecture 04 - I. Natural source Cells must be...

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