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)

Rijal, R., Consalvo, K. M., Lindsey, C. K. & Gomer, R. H. An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells. Mol. Biol. Cell 30, 242–255 (2019).

Pilling, D., Chinea, L. E., Consalvo, K. M. & Gomer, R. H. Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils. J. Immunol. 202, 239–248 (2019).

Smolkin, T. et al. Complexes of plexin-A4 and plexin-D1 convey semaphorin-3C signals to induce cytoskeletal collapse in the absence of neuropilins. J. Cell Sci. 131, (2018).

 

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 sora***** on human bladdercancer cells. Br. J. Pharmacol. v 160, pp 1690–1698.

 

Wang et al., 2007. Investigation of the immunosuppressive activity of arte****** 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)

Chen, L. et al. CSRP2 suppresses colorectal cancer progression via p130Cas/Rac1 axis-meditated ERK, PAK, and HIPPO signaling pathways. Theranostics 10, 11063–11079 (2020).

Liu, C. et al. Epigenetically upregulated GEFT-derived invasion and metastasis of rhabdomyosarcoma via epithelial mesenchymal transition promoted by the Rac1/Cdc42-PAK signalling pathway. EBioMedicine 50, 122–134 (2019).

Yang, H. et al. Cytotoxic Necrotizing Factor 1 Downregulates CD36 Transcription in Macrophages to Induce Inflammation During Acute Urinary Tract Infections. Front. Immunol. 9, 1987 (2018).

Tormos, A. M. et al. p38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging. PLoS One 12, e0171738 (2017).

Guo, Y. et al. Cytotoxic necrotizing factor 1 promotes prostate cancer progression through activating the Cdc42-PAK1 axis. J. Pathol. 243, 208–219 (2017).

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)

Shin, S. K. et al. Exogenous 8-hydroxydeoxyguanosine ameliorates liver fibrosis through the inhibition of Rac1-NADPH oxidase signaling. J. Gastroenterol. Hepatol. 35, 1078–1087 (2020).

Benz, P. M. et al. AKAP12 deficiency impairs VEGF-induced endothelial cell migration and sprouting. Acta Physiol. 228, (2020).

Thamilselvan, T. et al. P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells. Cell. Physiol. Biochem. 54, 1218–1230 (2020).

Yu, Q. et al. A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostate. J. Biol. Chem. 294, 12231–12248 (2019).

Borin, M. et al. Rac1 activation links tau hyperphosphorylation and Aß dysmetabolism in Alzheimer’s disease. Acta Neuropathol. Commun. 6, 1–17 (2018).

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)

Iweka, A. et al. The lipid phosphatase‐like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory molecules. J. Neurochem. jnc.15271 (2021) doi:10.1111/jnc.15271.

Zhou, Y. et al. Upregulation of ARHGAP30 attenuates pancreatic cancer progression by inactivating the β-catenin pathway. Cancer Cell Int. 20, 1–12 (2020).

Ramírez‐Ramírez, D. et al. Rac1 is necessary for capacitation and acrosome reaction in guinea pig spermatozoa. J. Cell. Biochem. 121, 2864–2876 (2020).

Guo, Y. et al. Cytotoxic necrotizing factor 1 promotes bladder cancer angiogenesis through activating RhoC. FASEB J. 34, 7927–7940 (2020).

Wang, R. et al. Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cells. Life Sci. 261, 118468 (2020).

Wee, K. et al. Snail induces epithelial cell extrusion by regulating RhoA contractile signalling and cell–matrix adhesion. J. Cell Sci. 133, (2020).

Barabutis, N. et al. Protective mechanism of the selective vaso****** V1A receptor agonist selepressin against endothelial barrier dysfunction. J. Pharmacol. Exp. Ther. 375, 286–295 (2020).

Wang, Q. et al. Thymol alleviates AGEs-induced podocyte injury by a pleiotropic effect via NF-κB-mediated by RhoA/ROCK signalling pathway. Cell Adh. Migr. 14, 42–56 (2020).

Kitchen, G. B. et al. The clock gene Bmal1 inhibits macrophage motility, phagocytosis, and impairs defense against pneumonia. Proc. Natl. Acad. Sci. U. S. A. 117, 1543–1551 (2020).

 
Tang, Jingshu et al. TIMP1 preserves the blood-brain barrier through interacting with CD63/integrin β 1 complex and regulating downstream FAK/RhoA signaling. Acta pharmaceutica Sinica. B vol. 10,6 (2020): 987-1003. doi:10.1016/j.apsb.2020.02.015
 
Moodley, Serisha et al. RET isoform-specific interaction with scaffold protein Ezrin promotes cell migration and chemotaxis in lung adenocarcinoma. Lung cancer (Amsterdam, Netherlands) vol. 142 (2020): 123-131. doi:10.1016/j.lungcan.2020.02.004
 

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)

Che, Guanghua et al. “Angiotensin II promotes podocyte injury by activating Arf6-Erk1/2-Nox4 signaling pathway.” PloS one vol. 15,3 e0229747. 2 Mar. 2020, doi:10.1371/journal.pone.0229747

Yu, Q. et al. A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostate. J. Biol. Chem. 294, 12231–12248 (2019).

Oga, T. et al. Genomic profiles of colorectal carcinoma with liver metastases and newly identified fusion genes. Cancer Sci. 110, 2973–2981 (2019).

Abdul-Salam, V. B. et al. CLIC4/Arf6 Pathway: A New Lead in BMPRII Inhibition in Pulmonary Hypertension. Circ. Res. 124, 52–65 (2019).

 

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. 

RIOS, A. et al. Participation of Rho, ROCK-2, and GAP activities during actin microfilament rearrangements in Entamoeba histolytica induced by fibronectin signaling. Cell Biol. Int. 32, 984–1000 (2008).

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. Arf1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK032-S
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  2. Arf6 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK033-S
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  3. Cdc42 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK034-S
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  4. Cdc42 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays BK034
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  5. PAK-PBD beads (binds active Rac/Cdc42 proteins) PAK02
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  6. Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays BK035-S
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  7. Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays BK035
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  8. 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
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  9. Ras Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays BK008
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  10. 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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  11. RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) BK036
    RhoA Activation Assay Biochem Kit (bead pull-down format) - 80 Assays
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  12. RhoA Pull-down Activation Assay Biochem Kit (bead pull-down format) BK036-S
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  13. Rhotekin-RBD beads (binds active Rho proteins) RT02
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