ou wish to study the gene in humans for the protein actin. You know that this pr
ID: 217700 • Letter: O
Question
ou wish to study the gene in humans for the protein actin. You know that this protein is made in cultured human fibroblast cells, but it is not the only protein made in the cells. You also know the amino acid sequence of human actin. At the time you are doing your experiment, no actin genes from other species have been isolated. You have available to you a human recombinant DNA library, cultured fibroblasts, purified antibodies specific for the protein actin, and all the supplies and equipment needed to perform the techniques that we discussed in class. Outline two different experimental approaches to isolate the actin gene from the recombinant library
Explanation / Answer
Approach 1
We are given the human recombinant DNA library.
This library is transformed into E. coli and the proteins are expressed and purified.
BY performing westernblot analysis using antibody specific to actin, the clone in the library that produces the actin protein can be identified.
Once this is done, the clone can be used to express the protein in higher concetration for subsequent analysis.
Approach 2-
We know that the actin protein is expressed in cultured fibroblast.
The fibroblast given to us are cultured in large amounts and lysed.
The cell lysate is passed on a column containg immobilized material with anti actin antibody.
After washing this column thorughly, we should expect pure actin to be bound to the antibody.
Using a high salt wash, only the actin can we eluted out of the column and used in follow up analysis.
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