2011 Week 3 Lab - BIO 122 FINAL0

2011 Week 3 Lab - BIO 122 FINAL0 - Stanford and Kabnick...

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Stanford and Kabnick Fall 2011 Bio 122: Week 3 Lab Goals : Bradford Assay: 1. To gain practice with conducting the Bradford Assay 2. To use a dilution series of BSA in a Bradford assay to generate a Standard Curve 3. To use Excel to plot Concentration v. Abs (@595nm) to create the linear equation. 4. To use the equation generated by the BSA Standard to determine the protein concentration of your cellular fractions for use in determining specific activity later on. SDH Assay: 1. To experience working with enzymes in the lab in order to gain a greater understanding of how enzymes work and how they are affected by a variety of factors 2. To gain experience in running an SDH assay 3. To collect the necessary data for a complete SDH trial of the mitochondrial fraction Preparing for Lab : With your group, research your assigned reagent, and develop a hypothesis for how your reagent might affect SDH activity. Your hypothesis assignment (one per group) must be submitted to your TA at the beginning of lab . See the website for the rubric (Week 2 and Week 3 folder). You will test your hypothesis during the Week 4 lab. Review the protocol for the Bradford Assay (below) o Note that the article describing the Bradford Assay is posted online, which will provide you with additional information about the assay. You can also review the materials posted for Recitation in Week 1, which describes this assay. Review the protocol for the SDH assay (below) o Be sure to know WHY each cuvette is being used in the assay. Notes :
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Stanford and Kabnick Fall 2011 Determining Protein Concentration of Liver Cell Fractions using a Bradford Assay 1. Creating your BSA curve: a. You will be given seven (7) tubes containing pre-diluted Bovine Serum Albumin to use for generating your standard Curve b. Set up 1 cuvette for each of the 7 concentrations in your curve. c. Vortex each tube of albumin thoroughly. Add 100µl of the appropriate standard to a corresponding cuvette. d. Add 3 ml Bradford’s Reagent into each cuvette. The total volume of the tubes will be 3.1 ml. e. Cover tubes with a small square of Parafilm and mix by inversion. f. Allow tubes to sit for 5 minutes inside the spectrophotometer with the frosted sides of the cuvette facing forwards (clear facing sideways to allow the light to pass through the cuvette). During the 5 minute wait you can start preparing your diluted samples (Step 2). g. Change the wave length on the spectrophotometer to read 595 nm by pressing the “Go to wl” button, pressing the clear button to remove the old wave length and then typing in 595. h.
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2011 Week 3 Lab - BIO 122 FINAL0 - Stanford and Kabnick...

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