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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Definitions:

Mode

The value appearing with the greatest frequency in a dataset.

Median

The middle value in a list of numbers sorted in ascending or descending order, or the average of the two middle numbers when the list has an even number of values.

Mathematical Average

The sum of a list of numbers divided by the count of numbers in the list, commonly referred to as the mean.

Median

The middle value in a data set when it is arranged in ascending or descending order, effectively dividing the data set in half.