Small G-protein activation assays measure the GTP-bound form of the protein from a cell or tissue extract. Cytoskeleton offers activation assays for Rho and Ras GTPase family members in two formats: the traditional pull-down assay and a G-LISA assay. The pull-downs utilize affinity beads linked to an effector protein that selectively binds the active GTPase which is then quantitated by Western blotting. The G-LISAs use a 96-well plate that has been coated with an effector protein to selectively bind the active GTPase, followed by quantitation with enzyme-linked immunosorbent assay (ELISA) techniques. Both assays are easy to use with distinct advantages for each.
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 tservice@cytoskeleton.com or 303-322-2254, ext 316.
GTPase Activation Assay Selection Guide
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Assay Factor |
Assay Considerations |
Recommendations |
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Assay principle |
Traditional Pull-Down
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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). |
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G-LISA
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Equipment |
Traditional Pull-Down
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Consider the equipment requirements prior to deciding upon an assay format.
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G-LISA
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Starting material required per assay |
Traditional Pull-Down 300 - 2000 µg per assay
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If starting material is limited, e.g. primary cells, the G-LISA assay is required. |
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G-LISA 5 – 50 µg per assay |
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Cost |
Traditional Pull-Down Available as:
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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.
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G-LISA Available in 96 well format. Each well can be broken off and used individually. |
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Sample handling |
Traditional Pull-Down Centrifugation steps and western blot analysis makes this assay most suitable for low sample numbers (≤10).
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<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. |
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G-LISA Amenable to high sample numbers (>10) or dose response/time-course analyses. Wells can be detached and used individually. |
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Quantification of results |
Traditional Pull-Down Western blot quantification has a very narrow linear range. Also, multiple manipulations of the samples result in a somewhat variable assay.
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G-LISA assays are recommended for simple, reproducible quantification of results. |
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G-LISA Provides a simple numeric readout |
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Reagents required that are not in the kits |
Traditional Pull-Down
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The G-LISA assay provides all reagents necessary to perform 96 activation assays. Wells can be detached and used individually.
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G-LISA
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