Description
For this protocol, you will be collecting fractions of buffer eluting from the column. Simultaneously, you will be
measuring the absorbance of each fraction to have a better understanding of what is eluting from the column.
Proteins and NADH absorb light at the wavelength we will be using; therefore, an increase in absorbance would
indicate that material (either wanted or unwanted) is eluting from the column.
For this assignment, you are you sketch a graph of what you would expect to see if you were to
graph adding the buffers vs absorbance of the eluent.
What is going into the column (sequentially) will be on the X axis, starting with your sample and ending with the
final wash. As these materials move through the column (and affinity chromatography magic happens), material
will elute OUT of the column. The absorbance you expect to see for this material will be on the Y axis. The Y axis
does not need specific numerical values (typical absorbance output is between 0 and 2 A). The X axis should be
labeled in mL and identify what was added and when. For example, if 5 mL A, 10 mL B then 25 mL C was added
sequentially to the column, the X axis should be ~40 mL in length. The first 5 mL being A, the next 10 being B and
the next 25 being C.
Your graph should contains peaks and valleys - Predict what is in these peaks based on the samples/buffers you
applied to the column.
Take a photo/scan of your graph and upload to the assignment.
