Cytoskeleton's Small G-protein Activation Assays measure the GTP-bound form of the protein from a cell or tissue extract.
This table outlines some important considerations when deciding which GTPase activation assay is right for you and summarizes what Cytoskeleton feels will be key decision points when choosing between pull-downs and G-LISAs as a means of measuring activation of small G-proteins. For more information and help with deciding which of Cytoskeleton’s activation assays are right for you, please check out the informational links on this page or contact our technical support team at firstname.lastname@example.org or 303-322-2254, ext 316.
Traditional pull-down assays use methods familiar to most labs and therefore provide a familiar point of entry.
The G-LISA assay is simple to use and has many advantages that should be considered (see below for comparisons).
Consider the equipment requirements prior to deciding upon an assay format.
Starting material required per assay
300 - 2000 µg per assay
If starting material is limited, e.g. primary cells, the G-LISA assay is required.
5 – 50 µg per assay
Cost issues to consider:
Cost per assay: The G-LISA is the best value per assay.
Cost per kit: The traditional pull-down assay starter kits generally provide the lowest cost per kit. NOTE: Consider the combo kit as a cost effective choice.
Available in 96 well format. Each well can be broken off and used individually.
Centrifugation steps and western blot analysis makes this assay most suitable for low sample numbers (≤10).
<10 samples: either assay
>10 samples: G-LISA recommended
NOTE: Dose responses and time courses are often recommended and will greatly increase the number of samples being analyzed.
Amenable to high sample numbers (>10) or dose response/time-course analyses. Wells can be detached and used individually.
Quantification of results
Western blot quantification has a very narrow linear range. Also, multiple manipulations of the samples result in a somewhat variable assay.
G-LISA assays are recommended for simple, reproducible quantification of results.
Provides a simple numeric readout
Reagents required that are not in the kits
The G-LISA assay provides all reagents necessary to perform 96 activation assays. Wells can be detached and used individually.
Cytoskeleton's Activation Assays have been cited hundreds of times over the past decade. A select few are described here, for more citations on individual products please use the "Citations" tab on each individual product page.
Howe and Addison, 2012. RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA. Vascular Cell. v 4, p 1.
Zhou et al., 2012. HSV-mediated gene transfer of C3 transferase inhibits Rho to promote axonal regeneration. Exp. Neurol. http://dx.doi.org/10.1016/j.expneurol.2012.06.016.
Khan et al. (2011). Geranylgeranyltransferase type I (GGTase-I) deficiency hyperactivates macrophages and induces erosive arthritis in mice. J Clin Invest doi:10.1172/JCI43758.
Yi H, Tao L, Feng TX, Ken C, Ming LL. (2010). Effects of ischemic preconditioning on vascular reactivity and calcium sensitivity after hemorrhagic shock and their relationship to the Rho A-Rho-kinase pathway in rats. J Cardiovasc Pharmacol.
Answer 1: Cytoskeleton, Inc. offers a large number of G-LISA and traditional pull-down activation assays to study the biology and biochemistry of small G-proteins. Our G-LISA activation assays provide an improved method of measuring the activity of small G-proteins utilizing a simple and quick protocol in 96-well format to provide extremely accurate results. To complement our G-LISA line of activation assays, we also offer the most efficiently designed and complete traditional pull-down activation assays available.
Based on enzyme-linked immunosorbent assay (ELISA) technology, our G-LISA activation assays utilize a 96 well format with 12 x 8 well strips that provide the flexibility to run 1 to 96 wells (each well is a condition/treatment) at one time. This flexibility is especially important if any concentration, dose or time course analyses will be performed. Pull-downs are limited to the number of wells in each gel (usually 10-15) that will be run. The G-LISA assays are also more accurate and quantitative than pull-downs with greater sensitivity while using less material per well. The isotype specificity depends on the antibodies being used to capture and visualize the small G-proteins. We have designed both types of activation assays to specifically target RhoA and Rac1. Each of these assays can be easily modified to study RhoB and RhoC or Rac2 and Rac3, respectively. We also offer activation assays for Cdc42, Ras, and RalA. For a quick “look and see” experiment with a few treatment conditions (one drug concentration and time point), pull-downs are convenient. Pull-downs also utilize methods that are very familiar to most labs and so offer a convenient entry point for small G-protein activation assays. Besides the standard sized pull-down kits (50-80 assays, depending on protein), we also offer a starter-sized kit (20 assays) for RhoA, Rac1, Cdc42, and Ras. To screen for multiple small G-proteins, we recommend the Combo starter pull-down kit which provides 10 assays each for RhoA, Rac1, and Cdc42.
For an interactive guide to help choose the activation assay that is right for you, click here.
Answer 2:Yes, the G-LISA activation assays are well-cited in the scientific literature. Please see the “citations” tab above for links to some current citations for our RhoA, Rac and Cdc42 G-LISA activation assays.