Pull-down based small G-protein activation assays work by using a domain of an effector protein coupled to agarose beads. These affinity beads only bind the active (GTP-bound) form of the protein, thereby isolating the active pool of target small g-protein from a complex cell or tissue homogenate. The isolated material is run on a Western blot which is probed with an antibody to a specific isotype e.g. RhoA. Cytoskeleton's Pull-down activation assays are compatible with mouse, rat and human tissues and cells.
Cytoskeleton's kits have many more reagents than other suppliers (see Contents section Manual in Documents tab), which allows you non-stop experimental operation at every point in the procedure. In addition we provide affinity beads alone for greater flexibility in experimental design, PAK-PBD for Rac and Cdc42 (Cat. # PAK02), and Rhotekin-RBD for Rho (Cat. # RT02). An image of the beads is shown to the left, you will notice the blue colored beads which allow easy identification of the pellet during the centrifuge based washing procedure. Cytoskeleton was first to introduce colored beads in 2004 in response to customers requests to better visualize the pellet.
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 the 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 shown below. More citations are available on individual product pages.
Ras Activation Assay Biochem Kit (bead pull down format) (Cat. # BK008)
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.
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 researcher wants to do a “quick look and see” experiment involving a limited number of conditions (1 time point and 1 drug concentration or treatment condition) with a limited number of samples. Also, some researchers are more comfortable with the traditional pull-downs or need to do some new pull-down assays to directly compare with previous pull-down data.
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.
