MCB110L_lecture20

MCB110L_lecture20 - MCB110L Lecture 20 04/01/11 Andreas...

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Unformatted text preview: MCB110L Lecture 20 04/01/11 Andreas Martin Malachite green phosphomolybdate green color ATPase assays in class: phosphate released by the ATPase is detected through phosphomolybdate/malachite green complex formation -> endpoint readout + alternative assay: coupling ATP regeneration to NADH consumption -> continuous readout Experiment 5.10: Stability assays goal: determine conditions that might stabilize the protein / retain its activity Protein storage conditions: Temperature:- best storage at T 4C in glass or polypropylene tubes - short-term storage at 4C - long-term in liquid N2 or at -20 to -80C, prevent freeze-thaw cycles - addition of glycerol or ethylene glycol: prevents freezing at -20C Protein concentration: - dilute solutions are more prone to inactivation (e.g. by binding to surfaces) -> add filler or carrier proteins (e.g. BSA), 1 - 5 mg/ml Protein storage conditions: molecular crowding in the cell Protein concentration: - dilute solutions are more prone to inactivation (e.g. by binding to surfaces) -> add filler or carrier proteins (e.g. BSA), 1 - 5 mg/ml Protein storage conditions: Additives: - Cryoprotectants (glycerol, ethylene glycol), 25-50%, prevent ice-crystal formation - protease inhibitors -> prevent proteolytic cleavage - anti-microbial agents, e.g. sodium azide NaN 3 , 0.02-0.05% - metal chelators, e.g. EDTA, 1-5 mM, prevent metal-induced oxidation - reducing agents, e.g. DTT, -ME, 1-5 mM, prevent Cys oxidation probes to monitor T-induced unfolding: activity absorbance CD NMR (fluorescence, strong T-dependence) G = H T S G = RT lnK Gibbs free energy: H = U + p V H = U + ( p V ) H = Q for p = const. enthalpy: S = k B ln = number of possible states S = Q T for T = const. entropy: Native Unfolded simple 2-state model for monomeric protein: T m : U/N = 1, G = 0 T m N U Example for T-induced unfolding followed by absorbance (RNase T1) 2-state (un)folding Protein stability as a function of temperature Calculation of thermodynamic constants from thermal denaturation curves G = H T S = RT ln K lnK = H T S RT = H RT + S...
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MCB110L_lecture20 - MCB110L Lecture 20 04/01/11 Andreas...

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