Ras Pull-down Kit

September 25, 2017

University of South Alabama
Cancer Biology
Graduate Student

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Company:

ThermoFisher

Product Name:

Active Ras Pull-Down and Detection Kit

Catalog Number:

16117

This kit was used to evaluate levels of active Ras in various cancer cell lines prior to screening the cell lines with novel compounds. This product was selected because it included everything needed for determining Ras levels, and it gave a thorough protocol that was easy to follow.

Experimental Design and Results Summary

Application

Affinity Pull-down and Western Blot

Starting Material

BxPC-3, HPAF-II, SW-1990, CFPAC-1 cell lines

Protocol Overview

Cells are lysed in a provided lysis buffer and protein concentration of sample is quantified using your protein assay of choice (not provided). Glutathione agarose beads are washed in provided spin columns. 500 micrograms of lysate are incubated with the washed glutathione agarose beads and 80 micrograms of GST-RAF1-RBD for 1 hour. Then, the beads are washed 3 times with centrifugation following each wash. Finally, the bound active Ras is eluted from the beads using a provided SDS sample buffer with a reducing agent added (not provided). Then perform a western blot with the samples and probe the membrane with the provided anti-Ras antibody.

Tips

Adjust cell lysate protein concentrations to be similar so that similar volumes are added to the spin column for the incubation with the beads and GST-RAF1-RBD. For the western blot, be sure block the membrane and incubate the antibody with 5% BSA. If milk is used, the blot will not be as clear and will have significantly more background.

Results Summary

The assay produces a western blot that accurately depicts the amount of active, GTP bound Ras in your cells of choice. The kit also provides GDP and non-hydrolyzable GTPgS that can be used to load Ras from your cells with either GDP or GTPgS. GDP and GTP-bound Ras can then be used as negative and positive controls for the pull-down assay.

DOI or PMID #

N/A

Additional Notes

N/A

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Summary

The Good

The assay is easy to perform, has a thorough protocol, and is forgiving.

The Bad

The kit is expensive if performing a lot of assays, and optimization will be needed if you wish to use less reagents.

The Bottom Line

This kit is an extremely valuable tool for studying all aspects of Ras biology and can be adapted to a variety of applications.

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