Pull-down Assays

Small G-protein pull-down activation assays utilize affinity beads linked to an effector protein that selectively binds the active GTPase, followed by Western blot quantitation with highly isotype specific antibodies. These assays are compatible with mouse, rat, and human tissue and cell lysates.  All reagents to perform a non-stop experiment are included. In addition, small G-protein affinity beads, antibodies, proteins, activators, and inhibitors are available for individual purchase.

Also Available: 
ELISA based Activation Assay formats (G-LISA).  To help you choose watch our Activation Assay Video.  

Many publications cite the use of Cytoskeleton's kits in the Materials and Methods section of papers. Usually the citation is associated with a particular result in the form of a graph or image that helps you, the authors, present your findings. This indicates the utility of the Kits to produce publication quality data in a short timeframe thus helping improve the productivity of your efforts. Example citations for pull-down assay kits are show below. More citations are available on individual product pages.


Ras Activation Assay Biochem Kit (bead pull down format) (Cat. # BK008)

Ciaglia E., et al. 2013. N6-isopentenyladenosine, an endogenous isoprenoid end product, directly affects cytotoxic and regulatory functions of human NK cells through FDPS modulation. J. Leukocyte Biol. doi: 10.1189/jlb.0413190 jlb.0413190.  

 

Stoppa et al., 2012. Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells. Apoptosis. v 17, pp 219-228.

 

Jiang et al., 2010. Activation of Rho GTPases in Smith–Lemli–Opitz syndrome: pathophysiological and clinical implications. Hum. Mol. Gen. v 19, pp 1347–1357.

 

Kowluru and Kowluru, 2007. Increased oxidative stress in diabetes regulates activation of a small molecular weight G-protein, H-Ras, in the retina. Mol. Vis. v 13, pp 602-10.

 

Kowluru, 2010. Role of matrix metalloproteinase-9 in the development of diabetic retinopathy and its regulation by H-Ras. Invest. Ophthalmol. Vis. Sci. v 51, pp 4320-4326. 

 

Lito et al., 2008. Evidence that sprouty 2 is necessary for sarcoma formationby H-Ras oncogene-transformed human fibroblasts. J. Biol. Chem. v 283, pp 2002-2009.

 

Rose et al., 2010. Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladdercancer cells. Br. J. Pharmacol. v 160, pp 1690–1698.

 

Wang et al., 2007. Investigation of the immunosuppressive activity of artemether on T-cell activation and proliferation. Br. J. Pharmacol. v 150, pp 652–661.


Cdc42 Activation Assay Biochem Kit (bead pull down format) (Cat. # BK034)

Kawther Abu-Elneel, Tomoyo Ochiishi, Miguel Medina, Monica Remedi, Laura Gastaldi, Alfredo Caceres, and Kenneth S. Kosik (2008). A delta-Catenin Signaling Pathway Leading to dendritic protrusions. J Biol Chem 283 ,32781-32791.

 

Nur-E-Kamal, A., Ahmed, I., Kamal, J., Schindler, M. and Meiners, S. (2005). Three dimensional nanofibrillar surfaces induce activation of Rac. Biochem. Biophys. Res. Commun. 331, 428-434.

 

Slice, L. W., Chiu, T. and Rozengurt, E. (2005). Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. 280, 1582-1593.

 

Tang, D. D., Zhang, W. and Gunst, S. J. (2005). The Adapter Protein CrkII Regulates Neuronal Wiskott-Aldrich Syndrome Protein, Actin Polymerization, and Tension Development during Contractile Stimulation of Smooth Muscle.  J. Biol. Chem. 280, 23380-23389.

 

Liu, X. F., Ishida, H., Raziuddin, R. and Miki, T. (2004). Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Cζ (PKCζ) and regulates PKCζ activity. Mol. Cell. Biol. 24, 6665-6675.

 

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

 

Tang, D. D. and Gunst, S. J. (2004). The small GTPase Cdc42 regulates actin polymerization and tension
development during contractile stimulation of smooth muscle. J. Biol. Chem. 279, 51722-51728.


Rac1 Activation Assay Biochem Kit  (bead pull down format) (Cat. # BK035)

Slice, L. W., Chiu, T. and Rozengurt, E. (2005). Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. 280, 1582-1593.

 

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

 

Yang, S. A., Carpenter, C. L. and Abrams, C. S. (2004). Rho and Rho-kinase mediate thrombin-induced phosphatidylinositol 4-phosphate 5-kinase trafficking in platelets. J. Biol. Chem. 279, 42331-42336.

 

Zhang, Y., Chen, K., Tu, Y. and Wu, C. (2004). Distinct roles of two structurally closely related focal adhesion
proteins,  α-parvins and β-parvins, in regulation of cell morphology and survival. J. Biol. Chem. 279, 41695-41705.

 

Chromy, B. A., Nowak, R. J., Lambert, M. P., Viola, K. L., Chang, L., Velasco, P. T., Jones, B. W., Fernandez,
S. J., Lacor, P. N., Horowitz, P. et al. (2003). Self-assembly of Aβ(1-42) into globular neurotoxins. Biochemistry 42, 12749-12760.

 

Quadri, S. K., Bhattacharjee, M., Parthasarathi, K., Tanita, T. and Bhattacharya, J. (2003). Endothelial barrier strengthening by activation of focal adhesion kinase. J. Biol. Chem. 278, 13342-13349.


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.

 

Pixley, F. J., Xiong, Y., Yu, R. Y., Sahai, E. A., Stanley, E. R. and Ye, B. H. (2005). BCL6 suppresses RhoA
activity to alter macrophage morphology and motility. J. Cell Sci. 118, 1873-1883.

 

Birukova, A. A., Liu, F., Garcia, J. G. and Verin, A. D. (2004). Protein kinase A attenuates endothelial cell barrier dysfunction induced by microtubule disassembly. Am. J. Physiol. 287, L86-93.

 

Cetin, S., Ford, H. R., Sysko, L. R., Agarwal, C., Wang, J., Neal, M. D., Baty, C., Apodaca, G. and Hackam, D. J. (2004). Endotoxin inhibits intestinal epithelial restitution through activation of Rho-GTPase and increased focal adhesions. J. Biol. Chem. 279, 24592-24600.

 

Orr, A. W., Pallero, M. A., Xiong, W. C. and Murphy-Ullrich, J. E. (2004). Thrombospondin induces RhoA inactivation through FAK-dependent signaling to stimulate focal adhesion disassembly. J. Biol. Chem. 279, 48983-48992.

 

Sasai, N., Nakazawa, Y., Haraguchi, T. and Sasai, Y. (2004). The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. 6, 741-748.

 

Setiadi, H. and McEver, R. P. (2003). Signal-dependent distribution of cell surface P-selectin in clathrin-coated pits affects leukocyte rolling under flow. J. Cell Biol. 163, 1385-1395.


Arf6 Activation Assay Biochem Kit (bead pull down format) (Cat. # BK033-S)

Berg-Larsen A., et al. 2013. Differential regulation of Rab GTPase expression in monocyte-derived dendritic cells upon lipopolysaccharide activation: A correlation to maturation-dependent functional properties. PLoS ONE. 8: e73538. 

Question 1:  Why does Cytoskeleton still provide pull-down assays when G-LISA technology is clearly superior?

Answer 1: At Cytoskeleton, we strive to be the one-stop provider for small G-protein activation assays, whether they are the traditional pull-down or the new G-LISA assays. The pull-down assays are useful when a researchers wants to do a "quick look and see" experiment involving a number of conditions (1 time point and 1 drug concentration or treatment condition) with a limited number of samples. 

 

Question 2: Why do I get doublet bands in some experiments and not in others?

Answer 2:  Sometimes researchers have reported that when using our Cdc42 or Rac1 pull-down activation assays (Cat. # BK034 and BK035, respectively), the GTPase signal appears as a doublet band. The most sensitive point for doublet formation is after resuspending the beads in sample buffer, and is caused by insufficient denaturation of
proteins prior to SDS-PAGE chromatography. The solution is to use double the amount of 2 x sample buffer i.e. 40 µl,
to resuspend the pellet, and to boil them for 1min. Samples can be stored at -20°C and analysed at a later date.  1 x Sample buffer is 63 mM Tris pH 6.8, 2% (w/v) SDS, 5% (v/v) β-mercaptoethanol, 10% (v/v) glycerol, 0.05% (w/v) bromophenol blue.

 

For more information, check out the manuals in the Document tab, or e-mail tservice@cytoskeleton.com for more in-depth questions.

  1. RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays BK030
    RhoA / Rac1 / Cdc42 Activation Assay Combo Biochem Kit (bead pull-down format) - 3 x 10 assays
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