Question

Scenario: you isolate a new drug that seems to be particularly effective at inhibiting a metabolic pathway in tumor cells. You want to do an experiment that compares global changes in the number and types of different proteins in cells that are treated with the compound vs. untreated cells. how can this be accomplished experimentally.

( upper division biochemistry lab)

Answer 1

Cancerous cells demonstrate extremely active metabolism since they require uninterrupted supply of energy for sustenance. These cells, thus, demonstrate a discrete type of metabolic protein profile which includes not only cytosolic proteins but also organeller proteins such as mitochondria. Thus, it remains important to understand the protein profiling of cancerous cells.

In order to investigate the different in protein profile of treated vs untreated cells, following set of experiments can be conducted:

1. Let the cells grow under normal conditions until they reach log phase of cell division.
2. Divide the cells into two parts, one will be treated will drug while the other will serve as control.
3. Treat the cells in respective set of groups with either the drug and the placebo/vehicle.
4. Allow the drug to incubate for a sufficient period of time, say 6-24 hours.
5. Wash the cells off the media and scrape out the cells from both groups.
6. Suspend the cells from each group in cell lysis medium on ice.
7. Isolate the whole protein and perform quantification assay for investigating total amount of protein isolated.
8. Subject the respective protein isolates to 2-dimensionl SDS PAGE analysis.
9. Examine the results for any change in location and intensity of the protein spots.
10. Analyse these spots by MS-MS spectroscopy to investigate the unique nature of the proteins.

In this manner, the exact number and type of proteins undergoing differential expression profile in the cancerous cells with and without drug treatment can be investigated.