Since 2001, Cytoskeleton has provided the scientific communitywith the most robust, accurate, and time-saving kits to measureSmall GTP-binding protein (SmG) activation. Along the way, wehave developed numerous versions for different SmGs, such as Rho, Rac, Arf1 & 6, Ras, and Ral. Also, the quantifiable GLISAversions enabled a new wave of more sensitive applications, e.g.measurement in limited primary cell numbers and Matrigel 3Dmatrix. We continue to develop and maintain these high standards,which allow you to produce the best results in the least amount oftime.SmGs are involved in regulating cell signaling pathways and impacta wide range of cellular processes, functions, and morphology.The Pulldown version of the assay uses affinity beads which areincubated with the extract and then separated by centrifugation.The pelleted products are separated by SDS-PAGE and blottedonto a membrane for western analysis of the SmG of interest. TheGLISA® format is a modified ELISA which has the affinity reagentpermanently attached to the well of a 96-well plate. The extract is incubated in the well which is then washed and probed withprimary and secondary antibodies.

User-Friendly Formats

Accurate and Sensitive

Over 500 Citations

 

Applications for G-LISAs 

Swiss 3T3 fibroblasts were treated with 1 μg/ml CN04 for the indicated times and cell lysates were subjected to G-LISA activation assays for RhoA (Cat.# BK124), Rac1 (Cat.# BK128), and Cdc42 (Cat.# BK127).

Swiss 3T3 fibroblasts were serum starved for 24h, then either treated with 10 ng/ml of EGF for 2 min (Lanes 4 & 5) or left untreated (Lanes 2 & 3).  Rac1 activation was measured using the Rac1 Activation pull-down assay (Cat. # BK035) (Lanes 2-5).  Lane 1 is 20 ng of recombinant Rac1-His protein.

 

Measure active GTPases in 3-D cell cultures

  • Pull-down assays require a prohibitive amount of cell lysate

Measure active GTPases in primary cell lysates

  • Pull-down assays require >20-fold more lysate

Increase accuracy of active GTPase quantitations

  • Pull-down assays are analyzed by semi-quantitative western blotting

Measure active GTPases in tissue lysates

  • Pull-down assays require >20-fold more lysate

Applications for Pull-downs

Measure active GTPase isoforms 

  • Active levels of multiple Ras- and Rho-subfamily GTPase isoforms can be detected

Complement G-LISA findings 

  • Use two different, validated techniques to quantitate active GTPase levels

Cited in hundreds of peer- reviewed articles.

3-D Cell Culture and G-LISAs: 

 

Keely P.J. et al. 2007. Investigating integrin regulation and signaling events in three-dimensional systems. Methods Enzymol. 426, 27-45. 

 

Ponik S.M. et al. 2013. RhoA is down-regulated at cell-cell contacts via p190RhoGAP-B in response to tensional homeostasis. Mol. Biol. Cell. 24, 1688-1699. 

 

Petroll W.M. et al. 2008. Dynamic assessment of fibroblast mechanical activity during Rac-induced cell spreading in 3-D culture. J. Cell Physiol. 217, 162-171. 

 

Primary Cells and G-LISAs: 

 

Valdez C.M. et al. 2016. The Rac-GAP alpha2-chimaerin regulates hippocampal dendrite and spine morphogenesis.  Mol. Cell. Neurosci. 75, 14-26. 


Nini L. and Dagino L. 2010. Accurate and reproducible measurements of RhoA activation in small samples of primary cells. Anal. Biochem. 398, 135-137. 


Flaiz C. et al. 2009. PAK kinase regulates Rac GTPase and is a potential target in human schwannomas. Exp. Neurol. 218, 137-144. 

 

Increased accuracy and G-LISAs:

 

Oliver A.W. et al. 2011. The HPV16 E6 binding protein Tip-1 interacts with ARHGEF16, which activates Cdc42. Br. J. Cancer. 104, 324 - 331. 

 

GTPase isoforms and Pull-downs:

 

Tabibian J.H. et al. 2014. Cholangiocyte senescence by way of N-Ras activation is a characteristic of primary sclerosing cholangitis. Hepatology. 59, 2263-2275. 


Kusuhara S. et al. 2012. Arhgef15 promotes retinal angiogenesis by mediating VEGF-induced Cdc42 activation and potentiating RhoJ inactivation in endothe­lial cells. PLoS ONE. 9:e45858. 

 

Pull-downs complement G-LISAs:

 

Tanaka T. et al. 2010. Monocyte chemoattractant protein-1/CC chemokine ligand 2 enhances apoptotic cell removal by macrophages through Rac1 activation. Biochem. Biophys. Res. Commun. 399, 677-682. 

 

Schlegel N. and Waschke J. 2009. VASP is involved in cAMP-mediated Rac 1 activation in microvascular endothelial cells. Am. J. Physiol. Cell Physiol. 296, C453-C462. 

 

Lai F.P.L. et al. 2009. Cortactin Promotes Migration and Platelet-derived Growth Factor-induced Actin Reorganization by Signaling to Rho-GTPases. Mol. Biol. Cell. 20, 3209-3223. 

 

Click here for more examples (see Citation tab).

 

 

 

cytoactiassay