Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Detergents – amphipathic/amphiphillic molecules that have a limited solubility and above a critical concentration form micelles (CMC).micelle monomerWhen micelles are present, there is always free monomer present tooIf detergent concentration is less than CMC, monomers are in solutionIf detergent concentration is above CMC, …..Detergents are typically broken down into 4 classes:detergent CMC (mmol) Aggregation # SDS 2.3 84 OG 25 27 CHAPS 8 11 LysoPC .007 186 Tween 80Tween 80 0.012 58**General Properties of Classes of Detergents for Membrane Protein Studies*SDS will help stabilize an all a-helix peptide/protein.Detergent Elimination Strategies:•Dialysis•Dilution•Size Exclusion•Biobeads (hydrophobic adsorption)Membrane Protein ReconstitutionSolubilizing MembranesProtein Purification SchemesFeatures of the 4 stages:...Ratio of detergent/lipid important, [] detergent importantTurbidity and Light Scattering:An increase in turbidity/light scattering is an indication of a larger size particle (as long as the wavelength used is larger than the particle size).The decrease is taken as solubilization.Turbidity is often measured in simple spectrophotometer ( 300-500nm)At solubilizing concentrations, most detergents will actually increase turbidity: shown to result from reassembly into multilammellar structures at point of solubilization.Turbidity and Light Scattering:“light scattering” performed as 90o scattered light under steady state conditions using a spectrofluorometer.It is more sensitive than turbidity, can have smaller sample sizes and go to longer wavelengths.“dynamic light scattering” = “quasielastic light scattering”=photon correlation spectroscopy: the intensity of scattered light is monitored in the ms time range.The Brownian motion of the particles induces a broadening of the spectrum, in a way that is related to their size and shape.You can determine round from disk from tubes etc.Entire Issue of BBA biomembranes:Volume 1508, Nov 2000Effects of Salt and UreaVesicle solubilizationSalt Effects on detergent CMCStudies go both ways: Solubilization of Liposomes by DetergentsRemoval of Detergents to Form
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