Biology 22, F09
Worksheet-2 : Ion exchange Purification of GFP and Cytochrome c (10 points)
This worksheet essentially requires you to analyze your data and interpret the results. Please follow
the directions outlined below and hand the results that are requested.
Objective: To analyze your data using a standard curve and to review the principles of separation using
ion exchange chromatography.
Format: Staple your work together using this sheet as the cover page with your name. Provide typed
responses to each of the questions. Responses will normally require 2-4 sentences. The graph can be done
by hand or by xcel, if you do the graph by hand, please present is neatly on graph paper appropriately
Due: September 29th (beginning of class)
GFP Data Presentation from this experiment: (you will only analyze the GFP results)
1. Please hand in a nicely presented table of your data, a) the absorbance values for your standard, b)
absorbance values of your samples and c) the concentration of your sample. If you needed to
dilute your sample, please be sure and include the dilution factor.
2. Provide a graph of your standard curve. Be sure the graph is properly labeled: the x and y axis
should be labeled and the graph should have a figure legend.
3. Hypothetically, if you had used gel filtration for this experiment, which molecule would have
eluted first and which last, and why?
4. What affect would you hypothesize 0.001M K+acetate have on GFP elution and why?
5. Thought questions: *Calculate your percent recovery of GFP from your sample mixture. The
mass recovered (mg)/mass added (mg) x 100.
How do you interpret your recovery? Did you recover a high percentage of your sample
from the original sample mixture (i.e. >90%)? Did you recover a low percentage, if so
why might your sample recovery be low or did your recover greater than 100% of your
sample and if so, what could be an explanation for this seemingly improbable
* Note: Assume that the 0.5 ml sample mixture which you added to column contained:0.2 mg/ml GFP.