Enzyme Kinetics III

Enzyme Kinetics III - Experiment 14: Isolation,...

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: , , Experiment 14 Isolation Purification and Kinetics of . E coli Alkaline Phosphatase Part 3 Biochemistry 332L - - 2 19 09 We will be completing our purification of the enzyme this week by . ( ) using chromatography DEAE diethyl aminoethyl cellulose is a , , chromatography resin that as the name implies has amino groups which , can combine with protons to become positively charged if the proton . , , concentration is high enough Or to say the same thing another way the resin will be positively charged if the pH is below the pK a of its amino . groups , For our alkaline phosphatase to stick to the column we want it to be . , negatively charged So do we want the pH to be above or below the , , isoelectric point pI? Satisfy yourself that the answer is above and review pH , pI and pK a . ( if necessary Also review column chromatography if you need .) to So we want to equilibrate the column with a buffer whose pH is above ( . ) the pI of alkaline phosphatase 4 5 and below the pKa of the DEAE amino ( ). groups 10 , When we are ready to elute the enzyme from the column we will , actually do so not by changing the pH to adjust charge but rather simply . by changing the ionic strength We will start by equilibrating the DEAE with ; , a very low ionic strength buffer this will take a fair amount of equilibration . so be patient and thorough when washing the column Then we will add the , , enzyme wash the column with the same buffer then elute with a buffer . - that is saltier The saltier buffer will interfere with the charge charge , attractions between the enzyme and DEAE causing the enzyme to come off . [ the column Note that the binding of alkaline phosphatase to DEAE is NOT , very tight binding so keep everything you elute from the column just in .] case the enzyme comes off when you are not expecting it to , . We have a lot to get done this lab period so work with facility Materials : Dialyzed Enzyme from Ammonium sulfate precipitation Stage III Enzyme - . / 5 mM Tris HCl pH 7 4 5 mM MgCl 2 Buffer A - . / 5 mM Tris HCl pH 7 4 5 mM MgCl 2 / 125mM NaCl Buffer B DEAE cellulose in Buffer A . - ( . ) 0 2M Tris HCl pH8 0 1mM p - - nitrophenyl β - D - ( ) ( . ) phosphate PNPP in Tris Buffer pH8 0 50μM - ( ) ( . ) nitrophenol PNP in Tris Buffer pH8 0 10N NaOH Bradford Reagent / 10 mM HEPES 5 mM MgCl 2 Dialysis Buffer
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Procedure 1. , ”. Recover your enzyme from the dialysis bag and label it Stage III enzyme Set aside 100μL for enzyme activity assay and 100μL for the protein . concentration analysis 2. . Take a buret and place a small amount of glass wool in the bottom Pour the . DEAE slurry into the column to make a resin bed about four to five cm high . , Leave the stopcock fully open while pouring the column Pour in stages so the resin settles as you go along rather than trying to pour the whole five cm at . . once A funnel might help 3. . , Use Buffer A to pack the column When the resin has settled wash down the
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Enzyme Kinetics III - Experiment 14: Isolation,...

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