Rfp lab: (PArt 1)
Overview:
2a, 4a, 5a, 6a, 6b
Part 2A:
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Materials:
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Part 4A:
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Part 5A:
Procedure:
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Part 6A:
Procedure:
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Part 6B:
Procedure:
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Lab 6:
Before the Lab:
1. How can solutions of different salt concentrations, which will unfold proteins to varying degrees, be used to help purify red fluorescent protein using column chromatography?
Proteins are unfolded by the salt concentrated buffer. The proteins that aren't washed out of the column refold when lower salt concentrations are added.
Lab 6 Questions:
1. Why is a protein’s conformation important for carrying out its function?
A protein's conformation determines the promoter regions, which are what gives each protein different functions.
2. What properties of the amino acids in a protein relate to protein folding?
The sequence of an amino acid.
3. Does the eluate containing your red fluorescent protein appear less bright or brighter than it did in the cell lysate following centrifugation? If there is a noticeable difference in the intensity of the red color, what might account for that?
The RFP-containg elute is brighter than the cell lysate, because it holds the most RFP.
4. What characteristic of red fluorescent protein is used as the basis for separation by column chromatography?
The red fluorescent protein sticks to the resin column when unfolded.
5. How might the column chromatography procedure be adjusted or modified to increase the purity of the red fluorescent protein sample?
The purity of the protein can be increased if we used more wash buffers and were more careful with collecting the protein.
1. How can solutions of different salt concentrations, which will unfold proteins to varying degrees, be used to help purify red fluorescent protein using column chromatography?
Proteins are unfolded by the salt concentrated buffer. The proteins that aren't washed out of the column refold when lower salt concentrations are added.
Lab 6 Questions:
1. Why is a protein’s conformation important for carrying out its function?
A protein's conformation determines the promoter regions, which are what gives each protein different functions.
2. What properties of the amino acids in a protein relate to protein folding?
The sequence of an amino acid.
3. Does the eluate containing your red fluorescent protein appear less bright or brighter than it did in the cell lysate following centrifugation? If there is a noticeable difference in the intensity of the red color, what might account for that?
The RFP-containg elute is brighter than the cell lysate, because it holds the most RFP.
4. What characteristic of red fluorescent protein is used as the basis for separation by column chromatography?
The red fluorescent protein sticks to the resin column when unfolded.
5. How might the column chromatography procedure be adjusted or modified to increase the purity of the red fluorescent protein sample?
The purity of the protein can be increased if we used more wash buffers and were more careful with collecting the protein.
Reflection and Summary
In this lab, we tried to make a colony of glowing red bacteria. While our bacteria did not glow, they did become the correct color , signifying that we managed to start production of the red protein. We did this by using restriction enzymes to isolate the RFP gene and then confirmed that our work was correct by using gel electrophoresis. Then, we used a process called chromatography to separate the protein from the bacteria.
In my opinion, this lab could have been better thought out and better organized. I know that a lot of my classmates thought that it was very long and confusing. It seemed to be rushed at some points, and drawn out at others. I, personally, liked my group for this project, which consisted of myself, Gabby, and Eric. I must admit that I and a little glad that our groups were assigned because I think that helped people be more productive. Over all, I enjoyed this lab, but it could definitely be improved by some more structure and instruction.
In my opinion, this lab could have been better thought out and better organized. I know that a lot of my classmates thought that it was very long and confusing. It seemed to be rushed at some points, and drawn out at others. I, personally, liked my group for this project, which consisted of myself, Gabby, and Eric. I must admit that I and a little glad that our groups were assigned because I think that helped people be more productive. Over all, I enjoyed this lab, but it could definitely be improved by some more structure and instruction.