Cellular and Molecular Biology

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Protein Isolation and Purification by GST Pull-Down


You work for a pharmaceutical company’s basic science division. Your lab has identified a small molecule inhibitor of your favorite protein (YFP). Your job now is to isolate this protein for structural studies.

You decide to recombinantly express YFP in bacteria for large scale isolation of the protein. Your supervisor suggests using a Glutathione-S-Transferase (GST) pulldown with TEV protease treatment.

The GST pulldown is a well-established method for protein purification but often generates false-positives.

How will you assay that the product of this pulldown is pure and what are the proper controls?


You perform the pulldown and follow with TEV treatment. As a control you withhold TEV treatment from one group. Afterwards you digest the protein with trypsin and submit your samples for mass spectrometry to ensure that the product is pure.


You perform the pulldown followed by TEV treatment. As a control you have a sample from bacteria which express GST without YFP. You assay purity by western blot with controls including whole cell lysate for GST alone and GST-YFP groups.


You perform the pulldown followed by TEV treatment. You will assay protein purity by flow cytometry. Since flow cytometry is internally controlled, there is no need for a separate control sample.


You perform the pulldown followed by TEV treatment. To assay protein purity you visualize your protein sample by transmission electron microscopy. Control groups include GST alone and GST-YFP without TEV treatment.