Enzyme Assay Using a standard curve to determine the concentration of enzyme in an unknown sample.

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Presentation transcript:

Enzyme Assay Using a standard curve to determine the concentration of enzyme in an unknown sample.

Examples of Standard Curves What is the X axis? What is the Y axis?

How can a curve be used? Predict unknown concentration when you test for the independent variable. “Assay”: analytic procedure to quantitatively measure the amount of activity in a sample. Use the results of the “assay” to predict unknown concentration.

Our Model System Catalase: You are working for a drug company and you want to make a “generic” of the enzyme based drug another company produces. You must determine how much of the active ingredient is present in the drug. Your research in the papers suggests the concentration is between .1 and 10% (mass (g)/volume (ml)) The closer to the exact amount you can come, the better your generic will work!

Model System What does catalase do? H2O2  H2O + O2 What can you “assay” for?

Methods and Materials Shared Materials: Individual Materials: Bottles of 6% and 3% H2O2 2 blenders (must be cleaned with soap at the end!) 2 electronic balances Individual Materials: ~25 g liver—source of catalase Conical tubes for reactions 2-disposable pipettes 2-1mL graduated pipettes Weigh boats 50 mL grad cylinder 250 mL beaker

Importance of variables & controls Could I design my own method to test the same IV and DV? FOCUS: What would I need to know to exactly repeat your methods?

Your job (Monday and Tuesday) Figure out the role of catalase in cell and why it is essential in cells. Design a standard curve to assay for the concentration of catalase in an unknown sample. Determine what type of best fit line fits your data. Estimate the concentration. Suggest a second run based on the information you found to further refine your value. Connect the methods for this lab to a real-world problem.

Is your curve usable? Is your curve linear? Curved? How do you know if the curve you’ve chosen is acceptable? Calculate “Y hat” This is the Y value for each X if the equation is used (rather than having your values be plotted) Residual = Yobserved-Yhat Graph residuals vs. X. If there is no pattern, then the regression line captures the overall pattern. http://www.youtube.com/watchv=6HcyewrPNEw