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

Examples of Standard CurvesWhat 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 SystemCatalase:

◦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 SystemWhat does catalase do?

◦H2O2 H2O + O2

◦What can you “assay” for?

Methods and MaterialsShared Materials:

◦Bottles of 6% and 3% H2O2

◦2 blenders (must be cleaned with soap at the end!)

◦2 electronic balancesIndividual 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 & controlsCould 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