You have a crude isolate of a new protein in yeast that you think might regulate
ID: 47218 • Letter: Y
Question
You have a crude isolate of a new protein in yeast that you think might regulate transcription. You first want to isolate and purify this protein from the mixture of other proteins.
a. Mention what techniques can be used to purify the protein. Please make sure you mention which technique will be appropriate for which kind of protein. What is the principle involved in the techniques you chose?
b. Now you have isolated this protein and you want to determine if this protein binds to DNA and or any other protein in the cell. Design an experiment to detect the following
Protein-protein interaction
Protein-DNA interaction
c. If it binds to DNA you also want to know which sequence it binds to. How will you identify the DNA sequence this protein binds to
d. You think it might be a transcriptional activator. Outline an experiment to identify it is a transcriptional activator.
Explanation / Answer
a. Protein purification methods:
Chromatography- this method exploits the physical properties of the protein or the element used in the solution.
Centrifugation – this method exploits the physical properties such density and size to separate proteins.
Gel electrophoresis – this method exploits the charge present on the protein to separate it.
b. To detect protein-protein interaction:
Western blotting
Develop target specific antibodies.
Separate the target protein using gel electrophoresis
After separation, stain the gel using antibodies specific to the target
Use secondary antibodies that are carrying reporter enzyme specific to primary antibodies
Expose it to the appropriate substrate
If the enzyme carrying antibodies bind to the primary antibodies we can observe the color
To detect protein-DNA interaction
Separate target DNA by agarose gel electrophoresis
Expose the gel with target protein
If the protein has the tendency to interact with the DNA, then they bound to the DNA
Identify the DNA bound protein complex by using the above mentioned method in western blotting.
c. Isolate the DNA-protein complex using polyacrylamide gel
Denature the protein using chemical or physical methods
Isolate the DNA
Denature the DNA, and expose it to the sequencing method
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