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If you have studied cellular movement or cell division, you have encountered Rho in the literature, because it regulates both processes. And the list of roles for Rho in the cell continues to grow! The prominence of Rho in the biology of non-diseased and diseased cells has caused researchers to continually optimize the Rho pull-down assay. This biochemical assay allows you to monitor the activity of Rho.
The principle that underlies this assay is that Rho is inactive in its GDP-bound form and active in its GTP-bound form. In response to stimuli, cells increase their Rho-GTP levels, which you can monitor via quantifying the level of Rho-GTP using an ELISA or Western blot.
What Is a Rho Pull-Down Assay?
A Rho pull-down assay follows the same general protocol as a co-immunoprecipitation as described by Ashleigh Miller’s Bitesize Bio article. However, in this assay, you “catch” Rho-GTP via the bait protein Rhotekin, which is a bona fide binding partner of Rho-GTP and contains a Rho binding domain (RBD).
Learn from My Experience with the Rho Pull-Down Assay
Most of my thesis work relied on this assay. When I first started, I found myself at the bottom of the learning curve. To compound the problem, I worked in a lab that didn’t validate their protocols with multiple people. Doing a literature search on this topic, I found that the protocol varies from lab-to-lab (and company-to-company). While there are similarities shared among protocols, fundamental features (like the buffer used to wash cells before lysing them) must be determined empirically. As a newbie, I quickly realized how reliant this assay was in the empirical data. I did gain experience using commercially available kits, but even they don’t guarantee a “one size fits all” solution either.
Years of experience later, I want to share with YOU the key features of a Rho pull-down assay that you must absolutely consider before embarking on the journey.
1. Test the Activity of Your RBD
Regardless of which purification protocol you use to purify RBD, make sure to test the activity of RBD before making aliquots. To do this, test different volumes of beads with a fixed volume of lysate treated with 0.1 mM GTP?S or 1 – 10 mM GDP to convert Rho into GTP and GDP-bound forms, respectively. Each round of purification that you do will call for this type of control experiment. This is because each purification will yield bait protein that may have less, more, or the same activity level as your previous purification.
2. Gentle with Your Cells
Gently lyse your cells! This tip is exactly what is said about conducting co-IPs, and it applies here too. There are many protocols out there, but there are at least two similarities: the addition of 10 mM magnesium chloride and protease inhibitors. Magnesium chloride is known to stabilize the GTP-bound Rho to prevent nucleotide exchange.
3. Minimize the Number of Samples
Minimize the number of samples that you process per assay. I learned this the hard way. Handling way too many samples will lead to irreproducible results. You will realize that strictly following incubation times is essential for consistent processing of samples. Deviation from indicated incubation times correlates with increased experimental variability. Incubation times should remain consistent from experiment to experiment. As a rule of thumb: six or seven samples per assay is manageable. Unless you’re The Flash, heed this warning!
I hope that I’ve allowed you to better ease your way into conducting Rho pull-down assays.
- Jaffe AB, Hall A. (2005) Rho GTPase: Biochemistry and Biology. Rev. Cell Dev. Biol. 21: 247–69.
- Ren XD, Schwartz MA. (2000) Determination of GTP loading on Rho. Methods Enzymol. 325: 264–72.
- Suryavanshi N, Ridley AJ. (2013) Determining Rho GTPase Activity by an Affinity-Precipitation Assay. Methods Mol. Biol. 1046: 191–202.