Useful Considerations When Choosing Your Assay Format

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