Pull-Down Assays Using Proteins That ONLY Bind to the GTP-Bound

question 30

Multiple Choice

Pull-down assays using proteins that ONLY bind to the GTP-bound version of the small G-protein can be used to detect when small G proteins like Rac1 have been activated by various growth factors.You were given two lysates,one having been treated with platelet-derived growth factor (PDGF) and the other not treated with PDGF.This method is similar to performing co-IPs,so based on that knowledge,which of the following steps is erroneous?

• A)To assay for levels of active Rac1,PAK1-beads (the protein that only binds GTP-bound Rac1-conjugated to beads similar to Nickel beads)are added to each of the lysates described above and incubated on a rocking platform (on ice).
• B)The tubes are vortexed well,loading buffer is added,and the samples are heated to aid in protein denaturation.
• C)The samples are loaded and analyzed by SDS-polyacrylamide gel electrophoresis,then the proteins are transferred to a membrane.
• D)The membrane (a.k.a.blot)will be probed with the anti-Rac antibody.

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