Topic%2005%20_%20Protein%20Purification%20_%20F10

Topic%2005%20_%20Protein%20Purification%20_%20F10 - Why...

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Why purify a protein? 1. To produce antibodies 2. To determine amino acid sequences 3. To create crystals for 3D structural analysis 4. To obtain info to clone genes 5a. To determine properties of natural proteins: - subunit composition, molecular weight, pI - for enzymes: K m , specific activity, turnover number, regulation 5b. To determine properties of recombinant proteins: - do recombinant proteins retain the same properties as those from natural sources? function “better”? function “differently”?
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Basic strategy for protein purification •Choice of tissue •Assay Identify and quantify the protein of interest • Method of homogenization •Fractionation High capacity / low selectivity methods to Low capacity / high selectivity methods • Assessment
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I. Choice of material, assay & homogenization • Choice of tissue, eg : – Interested in a species – Interested in a cell type – Interested in a particular protein – Source with a high abundance of the protein • Assay • Method of homogenization
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C O O C HO H CH 3 C O O C O CH 3 pyruvate L-lactate + NAD + NADH + H Exp 4: Purification of Lactate Dehydrogenase Choice of material: bovine muscle LDH can be approximately 5% of the total protein mass in a muscle cell
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Assay for the protein of interest Identify and quantify the protein of interest - enzymes: kinetic assay (enzyme assay) - substrates or products to be chromophores: use spectroscopy - if it is not an enzyme nor any chromophore available: - ELISAs detection by an antibody - affinity / binding assays often includes polyacrylamide gel analysis
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2 ways to assay LDH spectrophotometrically: + d[NADH] / dt appearance of product disadvantage: for LDH, the back reaction is favorable, more difficult to achieve linearity advantage: the change in substrate is easier to quantify accurately since the [P] = 0 M to start with OR – d[NADH] / dt disappearance of substrate advantage: for LDH, the back reaction is less favorable, easier to achieve linearity disadvantage: the change in substrate may not be significant enough to quantify accurately
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A coenzyme that carries reducing equivalents; commonly used in redox reactions Assay for LDH: Structures of NAD + and NADH
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A 260 nm 340 nm NAD + NADH NADH ε 340nm = 6220 M -1 cm -1 NAD + ε 340nm = 0.0 M -1 cm -1
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Lactate → Pyruvate direction Lac + NAD + Pyr + NADH + H + K eq 2.8 x 10 -6 = [Pyr][NADH] [Lac][NAD + ]
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Pyruvate → Lactate direction Pyr + NADH + H + → Lac + NAD + 400,000 = [Pyr][NADH] [Lac][NAD + ] K eq Assay in the Pyruvate to Lactate direction: – d[NADH] / dt less back reaction > achieve linear rates > rates α [LDH] > quantify LDH
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But … the initial [NADH] in the assay needs to have an A 340 < 1.5 at t o AND is this a sufficient concentration to achieve V max conditions?
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A = ε l c 1.5 = (6,220 M -1 cm -1 ) (1 cm) ([NADH]) [NADH] = 2.4 x 10 -4 M = 0.24 mM Is that high enough to insure Vo ~ Vmax conditions ? Ideally, want [NADH]
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Topic%2005%20_%20Protein%20Purification%20_%20F10 - Why...

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