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KeyPractical2_S2011 - MCB120L LAB PRACTICAL 2 SPRING 2011...

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MCB120L LAB PRACTICAL 2 SPRING 2011 Name:______ Answer Key Rvsd (All Labs) ____________ AP Sample#_________________ Lab Section (CIRCLE): Hilt/Dinesh-Kumar Lagarias Liu Morand You have been asked to determine the specific activity of an unknown sample of alkaline phosphatase. To do so, you have been given two vials: 2.0 ml of an unknown alkaline phosphatase solution in 50 mM enzyme assay buffer pH 9.5 (put on ice) and 2.0 ml 20 mM p-nitrophenylphosphate substrate (in water). In front of the lab are stock solutions of 0.1 M enzyme assay buffer pH 9.5, 0.5 mM p-nitrophenol (in water), 1.0 M K 2 HPO 4 Stop solution. Using microplate assay technology with the fixed time enzyme assay described below, determine the specific activity of your alkaline phosphatase preparation. Put your answers for the total protein and total enzyme content of your sample in the boxes on this page. Bradford Protein Assay Table/Protocol(s) used for protein measurements are attached and protein assay data is provided for you in the accompanying Microplate Data Table. Both assay protocols are based on those used in Experiments 2 and 4. Alkaline phosphatase assay. Use a 4 min fixed time microplate assay similar to that described in Exp. 3 with the following modifications. The assay mixture should consist of 125 μ L reaction volume containing 20 μ L enzyme in 50 mM enzyme assay buffer pH 9.5 and 2 mM p-nitrophenylphosphate substrate (final concentrations). After incubation for 4 min , sufficient 1.0 M K 2 HPO 4 Stop solution should be added to produce a final phosphate concentration of 0.375 M in each well and a final volume of 250 μ L. Total Enzyme Activity (2 ml) 0.786 ± 0.102 IU (EVEN SAMPLES) 0.390 ± 0.071 IU (ODD SAMPLES) Total Protein (2 ml) 3.05 ± 0.52 mg Unknown specific activity 0.258 IU/mg (EVEN SAMPLES) 0.128 IU/mg (ODD SAMPLES) Provide any and all notes, calculations, data, analyses, and conclusions in a clear, concise and organized manner ( ie any graphs or tables created must be properly constructed and labeled; all raw data must be appended, and properly labeled). Your write-up should be easy to read and follow - the reader should not have to make guesses or assumptions on what, why or how experimental protocols were performed, or make guesses or inferences for analyses and calculations; the reader does not have to flip pages back and forth looking for information. All graphs, tables and the write-up may be done on MS Excel or handwritten. There is no penalty for handwritten graphs, reports, etc; suitable graph paper is provided . ********************************************************************************** For extra credit: If pure alkaline phosphatase has a specific activity of 4.0 IU/mg, is your enzyme pure? Briefly justify your answer below. YES or NO (Circle answer)
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LP2 (S2011) Name:______ Answer Key (All Labs) ____________ Page #__ 2 __ Part I. Enzyme Assay Analysis Step 1. Write down materials, which include how you prepared dilutions Stock solutions (prepared by instructors) 2.0 ml of an unknown alkaline phosphatase solution in 50 mM unknown buffer (ON ICE) 0.1 M unknown buffer 20 mM p-nitrophenylphosphate (in water) 0.5 mM p-nitrophenol (in water) 1.0 M K 2 HPO 4 (prepare 4 mL 0.75 M K 2 HPO 4 by diluting 3 ml 1.0 M K 2 HPO 4 with 1 ml H 2 O)
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