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Unformatted text preview: : , , Experiment 13 Isolation Purification and Kinetics of . E coli Alkaline Phosphatase Part 2 Biochemistry 332L-- 2 12 09 , For our second week of the alkaline phosphatase project you ll be . purifying the Stage I enzyme prepared last week The alkaline phosphatase from . E coli is particularly thermostable , meaning it does not denature . readily when heated Although this is a property shared by many proteins- from bacteria that live in high temperature environments like hot springs the thermophilic- bacteria it is unusual among proteins from species like . E coli- which live in the temperature controlled environment of the . , , , mammalian gut Alkaline phosphatase however is relatively thermostable a . happy circumstance that allows us to purify it with a heating step By , , heating your Stage I enzyme any other proteins presumably not , . thermostable can be denatured and precipitated out , Once the heat purification is accomplished we will concentrate the . enzyme using ammonium sulfate precipitation Ammonium sulfate is more generally used in protein purification than in the use to which we are . , putting it Because of its very large ionic strength as the amonium sulfate , concentration in solution is increased charged and polar groups become . , more soluble while hydrophobic molecules become less so Put another way . the hydrophobic effect of the solution is enhanced This means that as the , ammonium sulfate concentration is increased hydrophobic molecules are more attracted to each other and the proteins will precipitate out in order of . their hydrophobicities The molecule with the most hydrophobic residues near , , . the surface of the molecule precipitates first then the next and so on If previous work has found the exact narrow ammonium sulfate concentration range over which a protein of interest goes from being soluble to being , insoluble this knowledge can be used to separate out that protein from a . , mixture of proteins However in this lab we will simply be using ammonium sulfate precipitation to concentrate the protein after the dilution it underwent . during the dialysis Materials Dialyzed Stage I Enzyme Powdered ammonium sulfate- ( . )/ 10mM Tris HCL pH7 4 10 mM MgSO 4 Dialysis Buffer 2M NaOH .- ( . ) 0 2M Tris HCl pH8 0 1mM p-- nitrophenyl - D- ( ) ( . ) phosphate PNPP in Tris Buffer pH8 0 50M p- ( ) ( . ) nitrophenol PNP in Tris Buffer pH8 0 , Polycarbonate Test Tubes 50 mL...
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