Small GTP-binding proteins such as RhoA, Rac1, and Cdc42 are involved in regulating cell signalling pathways and impact a wide range of cellular processes, functions, and morphology. They bind and hydrolyze GTP, thus being switched from the activated form to the inactivated form (Fig 1).
The most prominent family of small G proteins is represented by the Ras superfamily of proteins. The Rho subfamily belonging to this superfamily consists of proteins like RhoA, Rac1, and Cdd42. These proteins have been shown to be involved in the regulation of actin dynamics, thus playing a crucial role in processes like cell movement, intracellular transport, and organelle development. While RhoA affects actin stress fibers, Rac1 exhibits effects on lamellipodia and Cdc42 on filopodia.
2 methods to measure the activation of Small G proteins
Traditionally, small G activation has been measured with pull-down assays. Effector proteins which specifically bind to the activated forms of small G proteins and which are coupled to beads are used in these assays to pull-down the activated small G protein fraction from a cell lysate. Finally the results are obtained by Western Blot experiments (for typical results see Fig 2). Nevertheless, Cytoskeleton Inc. developed an alternative – the G-LISA technology (see typical results in Fig 3): in this assay format the effector proteins which selectively bind to activated small G proteins are coupled to 96 well plates, making it possible to run an ELISA-like activation assay.
How to choose the right assay format suited to your specific interest and your equipment?
To help you to choose the right kit for your project, I’ve summarized the points you’ll need to consider:
Whereas you need an SDS-PAGE apparatus and a Western blot apparatus for the traditional Pull-down assay, an Orbital Plate shaker, a Spectrophotometer (set to 490 nm), and a Multichannel pipette (recommended if handling high number of samples) are needed for the G-LISA technology.
Starting material required per assay
For the Pull-down assay you need 300-2000 µg per assay, but you only need 5-50 µg per assay for a G-LISA. Thus if the starting material is limited, e.g. primary cells, the G-LISA technology is recommended.
Number of samples
Whilst the number of samples which can be handled in parallel with the Pull-down assay is quite limited (app. 10), the G-LISA assay allows for handling of many more samples (96 well format).
As you end up with Western Blot results with the Pull-down assay, data obtained are semi-quantitative, whereas the G-LISA technology results in quantitative data.
You get data in 3 hours with the G-LISA, but a 2 day experiment is required for the Pull-down assay.
Price comparison per data point
The G-LISA technology offers the best value per assay, particularly if you anticipate performing >50 assays. If you are only planning to measure a few samples, the Pull-down assay (especially the starter kits and the combo kit) might be a good alternative if you can do without quantitative data.
Which small G proteins can be measured with these kits?
Pull-down assays are available for:
G-LISA assays are available for:
Need some help with choosing the right kit for your experiments? Contact me through the comments form below with your questions.
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