You wish to purify a human protein Explain the steps necessa

You wish to purify a human protein. Explain the steps necessary to purify this protein from a cell extract. Use the following terms in your description (but not necessarily in this order): digest DNA, PCR amplification, express protein, generate cDNA, run DNA on gel, chromatography, transform plasmid into bacteria, ligate DNA into plasmid, SDS-PAGE analysis, blue/white screening

Solution

Human proteins are present in very minute quantity in the cells. And it would be difficult to purify it from human cells, because the expression of proteins is different in different cells. Also, it would be difficult to take the human cells for experimentation, as its against the rules of ethics. So we would take the gene of that protein and express it in bacterial cell, as its easy to amplify a protein in a prokaryotic cell.

The steps for this experiment would be as follows:

1. DNA would be extracted from human cell and digested into smaller fragments with the help of nucleases. Desired gene fragment would be selected by running them on gel and selecting the appropriate band.
2. Desired gene would be selected and amplified by PCR technique.
3. After the desired gene is collected, its expressed in the bacterial cell by ligating it into the plasmid.
4. The bacterial cells would produce the proteins after incubation. We can extract the proteins by digesting the cells. After digestion, we can either use SDS PAGE and chromatography to purify and identify the desired protein.