Lab Practical Protocol

Lab Practical Protocol - Lab Practical Protocol A....

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Lab Practical Protocol A. Dilutions of Unknown in duplicate to be used for the enzyme and protein assays: 1:5, 1:25, 1:125 1. You will be given 0.5mL (500uL) of the unknown whose protein and enzyme concentration you must determine. 2. Proceed be preparing a set of serial dilutions. 3. Use a three step, five-fold dilution series. 4. Start by mixing 100uL of stock unknown with 400uL of phosphate buffer as your first step (a five fold dilution; 1:5) 5. Then, mix 100uL of the five fold dilution with 400uL more of water for your second step (1:25). 6. Then, mix 100uL of the twenty five fold dilution with 400uL more of water for your third step (1:125). 7. Label each tube with its dilution. 8. Do this entire procedure twice so that you can take average as necessary.
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B. Enzyme Assay: 2 X 1:125 dilutions -enzyme assays measure a dynamic property of your sample; therefore, timing is important in preparing for an enzyme assay, and final mixing must be done right at the spectrophotometer so measurements can be takes as soon as the reaction commences. -use macrocuvets 1. Obtain 2.5 mM pyruvate in a test tube. Keep the pyruvate solution at exactly room temperature, not colder (no ice), not warmer (do not hold in your hand). 2. In another test tube, obtain 50mM potassium phosphate for use as a blank. 3. Finally, obtain 0.9mM NADH. Cover it with Parafilm to minimize
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This note was uploaded on 07/08/2011 for the course BIOCHEM 153L taught by Professor Kim during the Summer '09 term at UCLA.

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Lab Practical Protocol - Lab Practical Protocol A....

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