2011 Week 4 Lab - BIO 122 FINAL v.2

2011 Week 4 Lab - BIO 122 FINAL v.2 - Stanford and Kabnick...

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Stanford and Kabnick Fall 2011 Bio 122: Week 4 Lab Goals : 1. To repeat the SDH assay using a second aliquot of the mitochondrial fraction 2. To test your group’s hypothesis about your assigned reagent Preparing for Lab : Review the protocol for the SDH assay (below) Review your hypothesis for how your assigned reagent or condition will affect SDH activity in this assay. Notes :
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Stanford and Kabnick Fall 2011 SDH Assay – Repeat Note: You can use the “2011 Bio 122 Lab Week 4 SDH Template” Excel file to record your data and to help with graphing. Two separate controls and a blank will be utilized: The blank contains Potassium Phosphate Buffer, Succinate and your undiluted extract. This assay reaction tube will allow you to zero out the turbidity present in your sample . The first control will be referred to as your no succinate control . This control contains potassium phosphate buffer, distilled water, undiluted extract and DCPIP (the artificial electron acceptor). This tube serves as a control for your reaction tubes. It enables you to say that all the activity you are observing occurs because of the extract . The extract contains your enzyme that catalyzes the oxidation of succinate to fumarate. The substrate, succinate, is absent from this tube. The second control will be referred to as your no enzyme control . This control contains potassium phosphate buffer, succinate and DCPIP and allows you to account for the turbidity of the sample in the presence of DCPIP . Each of these reaction mixtures will need to be made for each extract studied in the SDH assay. Procedure 1. Set up eight 4.5 ml cuvettes to be used in the assay. You cannot label these cuvettes so you must remember what tube is what- keep them in order! 2. Cut up eight squares of Parafilm that can cover the top of your cuvettes in later steps. 3. Add the appropriate amounts of Potassium Phosphate, Azide, and buffered sucrose from the list below to each cuvette. 4. Add the appropriate amount of extract to each cuvette. Read the table clearly and make sure you add the correct type of extract. MAKE SURE YOU USE THE UNDILUTED EXTRACT . 5. Add the appropriate amount of DCPIP to each cuvette. After adding the DCPIP make sure the solution is VERY navy blue in color. If not, show your TA, you may need to make adjustments to the volume of DCPIP added.
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