Lecture8 - MCB 110L Spring 2011 Lecture 8 2/11/2011 Qing...

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MCB 110L Spring 2011 Lecture 8 2/11/2011 Qing Zhong 306 Barker Hall
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Working with proteins • Proteins are not DNA - they are fragile Work fast, maintain sensible solution conditions (pH, stabilizers, etc.) Keep cold, solvated Avoid glass, frothing, vortexing (except when quantitating protein amount!) • Why? Held together by weak forces (VdW, H-bonds, etc.) Amino acid chain can be broken (base, proteases) At low concentrations, non-speciFc losses increase (adsorption)
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Cell
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Cell lysis - I • Considerations Source - organism, abundance, native or overexpressed, thermostable/fragile… Quantity: » 10’s-100’s μg - biochemistry » 1-100 mg - structure Purity - as high as needed Cost - raw material (e.g., tissue) vs. recombinant Natural inhibitors - available or not? (Example, nucleotide binding can stabilize a protein)
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Cell lysis - II • Process: Shred, shear, burst, squeeze source material Mechanical vs. enzymatic (see handout) • Goal: Force cells to release contents into defned medium (bu±±er, salts, inhibitors, etc.) • The method you use may be limited by protein characteristics: Low abundance - use recombinant expression (very
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Lecture8 - MCB 110L Spring 2011 Lecture 8 2/11/2011 Qing...

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