Name:

TF Name:

LS1a Fall 2014 Lab 3 Post-Lab Activity

Due date: Your post-lab assignment is due one week after completion of the lab at the start of your
next section.

1. (10 points) Attach a picture of your gel to your answers. On your picture, label each lane to
indicate which samples were loaded. Also label the molecular masses of the bands in lane with
your molecular weight marker.

2. (9 points) Describe how the color of the nickel-containing resin (Ni
2+
-resin) changed after adding
the crude lysate. Did the color of the resin change after washing the resin with the wash buffers?
What was the color of the eluate after you added the elution buffer?






3. (10 points) Nickel ions can bind either imidazole or 6xHis-tagged proteins to form an ion-dipole
complex. Imidazole and the 6xHis-protein compete for the same binding site on nickel, making
the formation of these two complexes mutually exclusive; therefore imidazole cannot bind to the
same nickel ion at the same time as the His-tagged protein. The coupled equilibria of nickel as it
binds either a 6xHis-tagged protein or imidazole is shown below:



Referring to the above reactions, briefly describe why including 300 mM imidazole in the elution
buffer allows it to remove 6xHis-GFP from the Ni
2+
-resin, but the wash buffers do not.






4. (11 points) Briefly comment on the results of your affinity purification as shown on your gel.
Overall, did the procedure lead to a successful purification of 6xHis-GFP from the crude lysate
sample that you started with? Are there any other proteins present in the elution sample? How
can you tell? Is 6xHis-GFP enriched in the elution sample compared to the crude lysate (input)
sample?



(Rxn 1)
(Rxn 2)

Name:

TF Name:

5. (10 points) The strength of the binding interaction between 6xHis-tagged proteins and Ni
2+
ions is
dependent on the pH of the solution in which the protein is dissolved. Would you expect the
strength of the binding interaction to change if the pH were lowered from pH 8 to pH 5? If so,
how would you expect it to change? Briefly explain your reasoning.






6. (10 points) GFPs fluorescence is the result of the three-dimensional structure of the protein,
which brings three amino acids physically close enough so that they can react to form the GFP
fluorophore (a fluorophore is a chemical entity that can absorb energy and re-emit it as light)
(see figure below):

(Figure 1)

This reaction occurs spontaneously after GFP is properly folded. The resulting fluorophore gives
GFP its fluorescent character. When the GFP fluorophore absorbs UV light, the fluorophore
achieves a higher energy state, and green light is released when the molecule relaxes to a lower
energy state. Details of the mechanism are shown below:

(Figure 2)

Identify the three amino acids in GFP that rearrange to form the GFP fluorophore. Write the
sequence in the direction from the amino-terminus to the carboxy-terminus.

EXCITED STATE