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Overview of Active GTPase Pull-Down and Detection Kits

Learn about the features of kits to measure expression of specific GTPases.

Active GTPase Pull-Down Kit Selection GuideThermo Scientific Pierce Active GTPase Pull-down and Detection Kits profile the activation of small GTPases by isolating them via their specific downstream effectors.

Active (GTP-bound) small GTPases are difficult to directly detect because no primary antibodies are available that are specific for the active forms. To circumvent this problem, the Pierce Kits precede detection with pull-down purification to differentiate active GTPases from their inactive, GDP-bound conformation based on their affinity for downstream effector proteins.

This affinity provides a method to distinguish the transient up- or down-regulation of the active populations of GTPases from global changes in GTPase expression. These transient changes in activation could be the experimental result of exposing cells to growth factors or other small-molecule stimulators or inhibitors, or due to changes that occur during cell growth, differentiation, tumorigenesis and metastasis.

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Active GTPase Pull-Down and Detection Kits

Active GTPase pull-down assay kits

Active GTPases interact with specific downstream effector proteins. These specific protein-protein interactions can be exploited to capture active GTPases by using the effector proteins as the bait in pull-down assays. The GTPase protein-binding domains (PBDs) of these effector proteins are expressed as functional GST-fusion proteins that can be immobilized to glutathione agarose, facilitating the capture and enrichment of the active forms of their cognate GTPases. Although effector proteins often interact with one or more active GTPase, each GTPase can be individually identified and the relative quantity determined by Western blot analysis after the pull-down assay.

The GTPase pull-down assay procedure is streamlined with the Thermo Scientific Pierce Active GTPase Pull-Down and Detection Kits, which are available for Arf1, Cdc42, Rac1, Rap1, Ras and Rho small GTPases. The pull-down affinity purification step is easily performed in convenient spin columns. After washing, the bound GTPase is recovered by eluting the GST-fusion protein from the glutathione resin. The purified GTPase is detected by Western blot using a specific antibody supplied in each kit. Relative differences in active GTPase signal can be quantified by densitometry or CCD camera. The GTPase pull-down procedure is already optimized for Western blot analysis using Thermo Scientific SuperSignal West Pico Chemiluminescent Substrate and HRP-conjugated secondary antibodies (available separately).

Kit Features:

  • Highly sensitive and accurate – reagents, antibodies and Western blot procedure ensure accurate controls and semi-quantitative results
  • Convenient – no need to express and purify your own GST-PBD fusion proteins or use expensive antibody-based affinity resins
  • Easy to use – achieve immediate success in a 2-hour assay
  • Efficient – spin columns separate liquid from resin to prevent sample loss and cross-contamination
  • Complete – kits include reagents for affinity purification, controls, cell lysis buffer and antibodies for Western blot detection
  • Validated – kits are functionally tested to ensure quality and performance
  • Compatible – kits work with a variety of cell types (e.g., mouse, rat and human)

 

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Active GTPase Pull-Down and Detection Kits

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Assay summary

Performing pull-down assays with the Active GTPase Pull-Down and Detection Kits is straightforward. Firstly, whole cell lysates are prepared from cultured cells using the lysis buffer provided in the kit. Lysate (containing active and inactive GTPase) is incubated with the GST-protein binding domain (PBD) fusion protein from the respective downstream effector protein and glutathione resin. This specific interaction enables isolation of the target active (GTP-bound) GTPase. Unbound lysate proteins, including inactive or GDP-bound GTPase, are removed using the spin columns. The active GTPase population is recovered from the glutathione resin using SDS-PAGE loading buffer and analyzed by Western blot.

Thermo Scientific Active GTPase Pull-Down and Detection Kit assay summary.

 

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Active GTPase Pull-Down and Detection Kits

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Measure activation of small GTPases via their specific downstream effectors

Menu of available kits

Menu of Active GTPase Pull-Down and Detection Kits. Protein names link to product pages.
Part No. GTPase Downstream effector binding domain-GST fustion Key cellular functions
16121 Arf1 GST-GGA3-PBD Assembly of coat proteins onto budding vesicles on trans-golgi network and endosomes
16119 Cdc42 GST-Pak1-PBD Filopodia, lamellipodia formation, and stress fibers
16118 Rac1 GST-Pak1-PBD Filopodia, lamellipodia formation, and stress fibers
16120 Rap1 GST-RalGDS-RBD Cell proliferenation/differentiation
16117 Ras GST-Raf1-RBD Cell proliferation/differentiation; motility
16116 Rho GST-Rhotekin-RBD Filopodia, lamellipodia formation, and stress fibers; motility
Each Active GTPase Pull-down and Detection Kit contains sufficient material to perform 30 pull-down assays from a 0.5 to 1mg cell lysate sample and the following components: GST-fusion protein with the GTPase-specific binding domain, glutathione agarose resin, GTPγS (positive control), GDP (negative control), lysis/binding/wash buffer, GTPase-specific primary antibody, SDS-PAGE sample loading buffer, spin columns and collection tubes.

 

GTPase assay comparison data

The effectiveness of the Active GTPase Pull-Down and Detection Kits is from a combination of the integrity of the GST-effector binding domain and the quality of the primary antibody provided in the kit. In a comparison of commercial assays available to detect active Ras, each product detects a similar activation pattern, but the Active GTPase Pull-down and Detection Kit is more sensitive than pull-down and ELISA kits from other suppliers.

Active Ras Pull-Down and Detection Kit is more sensitive than other commercially available kits
The Thermo Scientific Active Ras Pull-Down and Detection Kit is more sensitive than other commercially available kits. NIH3T3 cells were serum-starved and then stimulated with platelet-derived growth factor (PDGF) for the indicated times. Lysates were prepared and the amount of endogenous active Ras determined by pull-down, Western blot and densitometry (Panels A, B) or directly by ELISA (Panel C). Although similar trends in active Ras levels resulted, the Pierce Active Ras Pull-Down and Detection Kit (Panel A) had lower background on the Western blot (data not shown) and higher sensitivity than the other kits. All assays were performed according to the manufacturers' instructions.

 

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Active GTPase Pull-Down and Detection Kits

Applications

The following applications can be performed:

  • Follow the activation of a specific GTPase during cell differentiation
  • Determine the effects of small-molecule drugs on the activation of small GTPase pathways
  • Study the effects of small molecule inhibitors on GTPase activation
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Detection and localization of active GTPases in neuronal cell differentiation

References

  1. Bar-Sagi, D. and Hall, A. (2000). Ras and Rho GTPases: a family reunion. Cell 103:227-38.
  2. Taylor, S.J., et al. (2001). Nonradioactive determination of Ras-GTP levels using activated Ras interaction assay. Methods Enzymol 333:333-48.
  3. Benard V. and Bokoch, G.M. (2002). Assay of Cdc42, Rac, and Rho GTPase activation by affinity methods. Methods Enzymol 345:349-59.
  4. Charest, P. and Firtel, R. (2007). Big roles for small GTPases in the control of directed cell movement. Biochem J 401:377-90.
  5. Knox, A.L. and Brown, N.H. (2002). Rap1 GTPase regulation of adherens junction positioning and cell adhesion. Science 295:1285-8.
  6. Ren, X.-D. and Schwartz, M.A. (2000). Determination of GTP loading on Rho. Methods Enzymo. 325:264-72.
  7. Van Triest, M., et al. (2001). Measurement of GTP-bound Ras-like GTPases by activation-specific probes. Methods Enzymol. 333:343-8.
  8. Williams, D., et al. (2008). Rho GTPases and regulation of hematopoietic stem cell localization. Methods Enzymol 439:365-93.
  9. Yoon, H.Y., et al. (2005). In vitro assays of Arf1 interaction with GGA proteins. Methods Enzymol 404:316-32.

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Active GTPase Pull-Down and Detection Kits

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Measure activation of small GTPases via their specific downstream effectors

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