Activation Assays

Small G-protein Activation Assays measure the GTP-bound form of the protein from a cell or tissue extract.  Rho and Ras GTPase family member activation assays are available in two formats: the traditional pull-down bead format and our advanced ELISA based G-LISA format.  Watch our video or browse the options below to learn more about which format is right for you. 



G-LISA® is a registered trademark of Cytoskeleton, Inc (CO). All rights reserved.

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

Assay Factor

 

Assay Considerations

 

Recommendations

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).

 

G-LISA

  • 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).
 

Equipment

 

Traditional Pull-Down

  • SDS-PAGE apparatus
  • Western blot apparatus

 

 

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

 

 

 

G-LISA

  • 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.

 

G-LISA

5 – 50 µg per assay

 

Cost

 

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.

 

 

G-LISA

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.

 

G-LISA

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.

 

G-LISA

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.

 

 

 

G-LISA

  • 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

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.


G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)  (Cat. # BK124)

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.

RhoA Activation Assay Biochem Kit (bead pull down format) (Cat. # BK036)

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.

Question 1: Which is the best Small G-protein activation assay for my tissue? 

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.

Question 2: Are there any citations for the G-LISA series?

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.

Select from the categories below:

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  1. Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays BK035
    Rac1 Activation Assay Biochem Kit (bead pull-down format) - 50 Assays
    Learn More
  2. Rac1,2,3 G-LISA Activation Assay (Colorimetric format) 96 assays BK125
    G-LISA Rac 1,2,3 Activation Assay Biochem Kit (colorimetric format)
    Learn More
  3. Ras G-LISA Activation Assay Kit (Colorimetric Based) - 96 assays BK131
    G-LISA Ras Activation Assay Biochem Kit (Absorbance Based) Learn More
  4. Ras Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK008-S
    Ras Activation Assay Biochem Kit (bead pull-down format) - 20 Assays
    Learn More
  5. Ras Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays BK008
    Ras Activation Assay Biochem Kit (bead pull-down format) - 50 Assays
    Learn More
  6. RhoA G-LISA Activation Assay (Luminescence format) 96 assays BK121
    G-LISA RhoA Activation Assay Biochem Kit (luminescence format)
    Learn More
  7. RhoA G-LISA Activation Assay Kit (Colorimetric format) 24 assays BK124-S
    G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)
    Learn More
  8. RhoA G-LISA Activation Assay Kit (Colorimetric format) 96 assays BK124
    G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)
    Learn More
  9. RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK036-S
    RhoA Activation Assay Biochem Kit (bead pull-down format) - 20 Assays
    Learn More
  10. RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) - 80 Assays BK036
    RhoA Activation Assay Biochem Kit (bead pull-down format) - 80 Assays
    Learn More
  11. Signal-Seeker™ Acetyl-Lysine Detection Kit (10 assay) BK163-S
    Signal Seeker Acetylation Detection Kit (10 assays, immunoprecipitation format) Learn More
  12. Signal-Seeker™ Acetyl-Lysine Detection Kit (30 assay) BK163
    Signal Seeker Acetylation Detection Kit (30 assays, immunoprecipitation format) Learn More
  13. Signal-Seeker™ Phosphotyrosine Detection Kit (10 assay) BK160-S
    Signal Seeker Phosphotyrosine Detection Kit (10 assays, immunoprecipitation format) Learn More
  14. Signal-Seeker™ Phosphotyrosine Detection Kit (30 assay) BK160
    Signal Seeker Phosphotyrosine Detection Kit (30 assays, immunoprecipitation format)
    Learn More
  15. Signal-Seeker™ SUMOylation 2/3 Detection Kit (10 assay) BK162-S
    Signal Seeker SUMOylation 2/3 Detection Kit (10 assays, immunoprecipitation format) Learn More
  16. Signal-Seeker™ SUMOylation 2/3 Detection Kit (30 assay) BK162
    Signal Seeker SUMOylation 2/3 Detection Kit (30 assays, immunoprecipitation format) Learn More
  17. Signal-Seeker™ Ubiquitination Detection Kit (10 assay) BK161-S
    Signal-Seeker Ubquitination Detection Kit (10 assays, immunoprecipitation format) Learn More
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