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 or 303-322-2254, ext 316.  


GTPase Activation Assay Selection Guide

Assay Factor


Assay Considerations



Assay principle


Traditional Pull-Down

  • Utilizes affinity beads coated with an effector protein to selectively bind the active GTPase.
  • The signal is analyzed by western blot. 



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).



  • Uses a 96 well plate coated with an effector protein to selectively bind the active GTPase.
  • The signal is analyzed by enzyme-linked immunosorbent assay (ELISA).



Traditional Pull-Down

  • SDS-PAGE apparatus
  • Western blot apparatus



Consider the equipment requirements prior to deciding upon an assay format.





  • Orbital plate shakers (2)
  • Spectrophotometer (set to 490 nm)
  • Multichannel pipette (recommended if handling high number of sample)

Starting material required per assay


Traditional Pull-Down

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




Traditional Pull-Down

Available as:

  • Combo kit (10 assays each RhoA/Rac1/Cdc42)
  • Starter kit (20 assays)
  • Regular kit (50-80 assays depending on target protein)



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.


Sample handling


Traditional Pull-Down

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


Traditional Pull-Down

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


Traditional Pull-Down

  • Cell or tissue lysate
  • Protein quantitation reagent
  • Anti-mouse secondary antibody
  • SDS-PAGE buffers
  • SDS-PAGE gels
  • Molecular weight markers
  • Western blot buffers
  • Western blot detection reagents



The G-LISA assay provides all reagents necessary to perform 96 activation assays. Wells can be detached and used individually.





  • Cell or tissue lysate


For more detailed information pertaining to Cytoskeletons Activation Assays, please click on the links below 

More about traditional pull-down activation assays

More about G-LISA activation assays