G-LISA Assays

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Many publications cite the use of Cytoskeleton's G-LISA kits in the Materials and Methods section. Usually the citation is associated with a particular result in the form of a graph or image that helps the authors present their 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 G-LISA assay kits are shown below.  More citations are available on individual product pages.

 

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G-LISA RhoA Activation Assay Biochem Kit (luminescence format) (Cat. # BK121)

 

Nobe et al., 2012. Two distinct dysfunctions in diabetic mouse mesenteric artery contraction are caused by changes in the Rho A–Rho kinase signaling pathway. E. J. Pharmacol. v 683, pp 217-225.

 

Wang et al., 2012. RhoA/ROCK-dependent moesin phosphorylation regulates AGE-induced endothelial cellular response. Cardiovascular Diabetology. v 11, p 7.

 

Zuo et al., 2012. Cdc42 negatively regulates intrinsic migration of highly aggressive breast cancer cells. J. Cell. Physiol. v 227, pp 1399-1407.

 

Alvarez et al. (2010). Failure of Bay K 8644 to induce RhoA kinase-dependent calcium sensitization in rabbit blood vessels. British J of Pharmacology 160 ,1326-37.

 

Heckman-Stoddard et al. (2009). Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research 11 ,http://breast-cancer-research.com/content/11/4/R61.

 

Hammar et al. (2009). Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function. Endocrinology 150 ,2072-2079.

 

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Kinoshita et al., 2008. Mol Biol Cell. 19, 2289

 

Moniz et al. (2008). WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cellular Signalling 20 ,1762-68.

 

Lesato et al. (2008). Tiotropium Bromide Attenuates Respiratory Syncytial Virus Replication in Epithelial Cells. Respiration 76 ,434-441.

 

Kinoshita et al. (2008). Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation. Molecular Biology of the Cell 19 ,2289-2299.

 

Scott et al., 2007. J Invest Dermatol. v 127, p 668.

 

Schreibelt et al. (2007). Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB Journal 21 ,3666-3676.

 

Tanaka et al.  (2007). Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth. J. Biol. Chem 282 ,10506-10515.

 

Higashibata et al., 2006. BMC Biochem. 7, 19

 

Zuo et al., 2006. Biochem Biophys Res Commun. 351, 361

 

Woods and Beier, 2006. J Biol Chem. 281, 13134

 

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G-LISA RhoA Activation Assay Biochem Kit (colorimetric format)  (Cat. # BK124)

 

Howe and Addison, 2012. RhoB controls endothelial cell morphogenesis in part via negative regulation of RhoA. Vascular Cell. v 4, p 1.

 

Yang and Kim, 2012. The RhoA-ROCK-PTEN pathway as a molecular switch for anchorage dependent cell behavior. Biomaterials. v 33, pp 2902-2915.

 

Garrido-Gomez et al., 2012. Annexin A2 is critical for embryo adhesiveness to the human endometrium by RhoA activation through F-actin regulation. FASEB J. doi: 10.1096/fj.12-204008.

 

Greco et al., 2012. Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor. Cardiovasc Res. doi: 10.1093/cvr/cvs204.

 

Chen et al., 2012. Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR. Biochem. Pharmacol. v 83, pp 1183-1194. 

 

Zhou et al., 2012. HSV-mediated gene transfer of C3 transferase inhibits Rho to promote axonal regeneration. Exp. Neurol. http://dx.doi.org/10.1016/j.expneurol.2012.06.016.

 

McCoy et al., 2012. Protease-activated receptor 1 (PAR1) coupling to Gq/11 but not to Gi/o or G12/13 is mediated by discrete amino acids within the receptor second intracellular loop. Cellular Signalling. v 24, pp 1351-1360.

 

Ramseyer et al., 2012. Tumor Necrosis Factor α Decreases Nitric Oxide Synthase Type 3 Expression Primarily via Rho/Rho Kinase in the Thick Ascending Limb. Hypertension. v 59, pp 1145-1150.  

 

Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases. PLoS ONE. v 7, e35046. 

 

Jin et al. (2011). Increased SRF Transcriptional Activity is a Novel Signature of Insulin Resistance in Humans and Mice. J Clin Invest.

 

Ganguly et al. (2011). Adiponectin Increases LPL Activity via RhoA/ROCK-Mediated Actin Remodelling in Adult Rat Cardiomyocytes. Endocrinology 152 ,247.

 

Rapier et al., 2010. Cancer Cell Int. 10, 24

 

Nini L, Dagnino L. (2010). Accurate and reproducible measurements of RhoA activation in small samples of primary cells. Anal Biochem 398 ,135-7.

 

Yang et al. (2010). Fluoride induces vascular contraction through activation of RhoA/Rho kinase pathway in isolated rat aortas. Environmental Toxicology and Pharmacology 29 ,290-296.

 

Musso et al. (2010). Relevance of the mevalonate biosynthetic pathway in the regulation of bone marrow mesenchymal stromal cell-mediated effects on T cell proliferation and B cell survival. Haematologica DOI: 10.3324/haematol.2010.031633.

Lichtenstein et al. (2010). Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives. J Alz Dis 22 ,1135.

Ridgway et al. (2010). Modulation of GEF-H1 Induced Signaling by Heparanase in Brain Metastatic Melanoma Cells. J Cellular Biochemistry 111 ,1299-1309.

 

Fang et al. (2010). Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1. J Biol Chem 285 ,26211-26222.

 

Romero et al. (2010). Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture. Toxicol. Sci. 118 ,602-612.

 

Rapier et al. (2010). The extracellular matrix microtopography drives critical changes in cellular motility and Rho A activity in colon cancer cells. Cancer Cell International 10 ,24.

Hammar et al. (2009). Role of the Rho-ROCK (Rho-Associated Kinase) Signaling Pathway in the Regulation of Pancreatic β-Cell Function. Endocrinology 150 ,2072-2079.

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Ramirez et al., 2008. J Immunol. 180, 1854

 

Sequeira et al. (2008). Rho GTPases in PC-3 prostate cancer cell morphology, invasion and tumor cell diapedesis. Clinical and Experimental Metastatis 25 ,569-579.

 

Moore et al. (2008). Rho inhibition recruits DCC to the neuronal plasma membrane and enhances axon chemoattraction to netrin 1. Development 135 ,2855-2864.

 

Kinoshita et al. (2008). Apical Accumulation of Rho in the Neural Plate Is Important for Neural Plate Cell Shape Change and Neural Tube Formation. Molecular Biology of the Cell 19 ,2289-2299.

 

Seifert et al. (2008). Differential activation of Rac1 and RhoA in neuroblastoma cell fractions. Neurosci Lett 450 ,176-180.

 

Korobova and Svitkina  (2008). Arp2/3 Complex Is Important for Filopodia Formation, Growth Cone Motility, and Neuritogenesis in Neuronal Cells. Mol. Biol. Cell. 19 ,1561-1574.

 

Mercer and Helenius (2008). Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 320 ,531.

 

Keely et al., 2007. Methods Enzymol. v 426, p 27.

 

Scott et al., 2007. J Invest Dermatol. v 127, p 668.

 

 Schreibelt et al. (2007). Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase, and PKB signaling. FASEB Journal 21 ,3666-3676.

Tanaka et al.  (2007). Neural Expression of G Protein-coupled Receptors GPR3, GPR6, and GPR12 Up-regulates Cyclic AMP Levels and Promotes Neurite Outgrowth. J. Biol. Chem 282 ,10506-10515.

 

Bradley et al., 2006. Mol Biol Cell. 17, 4827

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G-LISA Rac 1,2,3 Activation Assay Biochem Kit (colorimetric format) (Cat. # BK125)

 

Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases. PLoS ONE. v 7, e35046. 

 

Halpert et al. (2011). Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica. Protoplasma DOI: 10.1007/s00709-010-0254-1.

 

Vives et al. (2011). The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. Journal of Bone and Mineral Research 26 ,1099.

 

Tanaka et al., 2010. Biochem Biophys Res Commun. v 399, p 677.

 

Johanna et al. (2010). Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia. Neurochem International 57 ,762-773.

 

Lichtenstein et al. (2010). Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives. J Alz Dis 22 ,1135.

 

Ridgway et al. (2010). Modulation of GEF-H1 Induced Signaling by Heparanase in Brain Metastatic Melanoma Cells. J Cellular Biochemistry 111 ,1299-1309.

 

Fang et al. (2010). Allogeneic Human Mesenchymal Stem Cells Restore Epithelial Protein Permeability in Cultured Human Alveolar Type II Cells by Secretion of Angiopoietin-1. J Biol Chem 285, 26211-26222.

 

Romero et al. (2010). Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture. Toxicol. Sci. 118 ,602-612.

 

Baumer et al., 2009. J Cell Physiol. 220, 716

 

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

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.

 

Mercer and Helenius (2008). Vaccinia Virus Uses Macropinocytosis and Apoptotic Mimicry to Enter Host Cells. Science 320 ,531.

 

Pontow et al., 2007. Virology. 368, 1

 

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G-LISA Rac 1 Activation Assay Biochem Kit (luminescence format)  (Cat. # BK126)

 

Binker et al., 2011. TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2. Biochem. Biophys. Res. Comm. v 405, pp 140-145.

 

Sayedyahossein et al., 2012. Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes. FASEB J. doi: 10.1096/fj.12-207852.

 

Kikuchi et al., 2012. Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma. Oncogene. doi:10.1038/onc.2012.46.

 

Eggers et al., 2012. STE20-related Kinase Adaptor Protein α (STRADα) Regulates Cell Polarity and Invasion through PAK1 Signaling in LKB1-null Cells. J. Biol. Chem. v 287, pp 18758-18768.

 

Vives et al. (2011). The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. Journal of Bone and Mineral Research 26 ,1099.

 

McHenry et al. (2010). P190B RhoGAP has pro-tumorigenic functions during MMTV-Neu mammary tumorigenesis and metastasis. Breast Cancer Res.

 

Johanna et al. (2010). Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia. Neurochem International 57 ,762-773.

 

Baumer et al., 2009. J Cell Physiol. 220, 716

 

Heckman-Stoddard et al. (2009). Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research 11 ,http://breast-cancer-research.com/content/11/4/R61.

 

Chastre et al. (2009). TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness. FASEB Journal 23 ,916–928.

 

Ramirez et al., 2008. J Immunol. 180, 1854

 

Moniz et al. (2008). WNK2 modulates MEK1 activity through the Rho GTPase pathway. Cellular Signalling 20 ,1762-68.

 

Pontow et al., 2007. Virology. 368, 1

 

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G-LISA Cdc42 Activation Assay Biochem Kit (colorimetric format)  (Cat. # BK127)

 

Chen et al., 2012. Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR. Biochem. Pharmacol. v 83, pp 1183-1194. 

 

Eggers et al., 2012. STE20-related Kinase Adaptor Protein α (STRADα) Regulates Cell Polarity and Invasion through PAK1 Signaling in LKB1-null Cells. J. Biol. Chem. v 287, pp 18758-18768.

 

Dhaliwal et al., 2012. Cellular cytoskeleton dynamics modulates non-viral gene delivery through RhoGTPases. PLoS ONE. v 7, e35046. 

 

Oliver et al., 2011. Br J Cancer. v 104, p 324.

 

McHenry et al. (2010). P190B RhoGAP has pro-tumorigenic functions during MMTV-Neu mammary tumorigenesis and metastasis. Breast Cancer Res.

 

Lichtenstein et al. (2010). Secretase-Independent and RhoGTPase/PAK/ERK-Dependent Regulation of Cytoskeleton Dynamics in Astrocytes by NSAIDs and Derivatives. J Alz Dis 22 ,1135.

 

Schlegel and Waschke (2010). Impaired cAMP and Rac 1 Signaling Contribute to TNF-α-induced Endothelial Barrier Breakdown in Microvascular Endothelium. Microcirculation 16 ,521.

 

Stankiewicz et al. (2010). GTPase activating protein function of p85 facilitates uptake and recycling of the β1 integrin. Biochemical and Biophysical Research Communications 391 ,443.

 

Romero et al. (2010). Chronic Ethanol Exposure Alters the Levels, Assembly, and Cellular Organization of the Actin Cytoskeleton and Microtubules in Hippocampal Neurons in Primary Culture. Toxicol. Sci. 118 ,602-612.

 

Heckman-Stoddard et al. (2009). Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression. Breast Cancer Research 11 ,http://breast-cancer-research.com/content/11/4/R61.

 

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Rac1 G-LISA™ Activation Assay (Colorimetric Based)  (Cat. # BK128)

 

Antonov et al., 2012. Regulation of endothelial barrier function by TGF-β type I receptor ALK5: Potential role of contractile mechanisms and heat shock protein 90. J. Cell. Physiol. v 227, pp 759-771. 

 

Greco et al., 2012. Chemotactic effect of prorenin on human aortic smooth muscle cells: a novel function of the (pro)renin receptor. Cardiovasc Res. doi: 10.1093/cvr/cvs204.

 

Oshikawa et al., 2012. Novel role of p66Shc in ROS-dependent VEGF signaling and angiogenesis in endothelial cells. Am. J. Physiol. Heart Circ. Physiol. v 302, pp H724-H732.

 

Chen et al., 2012. Inhibition of tumor cell growth, proliferation and migration by X-387, a novel active-site inhibitor of mTOR. Biochem. Pharmacol. v 83, pp 1183-1194. 

 

Montalvo-Ortiz et al., 2012. Characterization of EHop-016, a novel small molecule inhibitor of Rac GTPase. J. Biol. Chem. v 287, pp 13228-13238.

 

Stefanini et al., 2012. Rap1-Rac1 Circuits Potentiate Platelet Activation. Arterioscler Thromb Vasc Biol. v 32, pp 434–441.

 

Vives et al. (2011). The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts. Journal of Bone and Mineral Research 26 ,1099.

 

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Ras G-LISA™ Activation Assay (Colorimetric Based)  (Cat. # BK131)

 

Gil-Henn et al., 2012. Arg/Abl2 promotes invasion and attenuates proliferation of breast cancer in vivo. Oncogene. doi:10.1038/onc.2012.284

 

Signal Amplification and Optimization Question 1: How can I increase the difference in Rho activation levels between control samples and stimulated samples? Question 2: How can I eliminate high variability between samples when using the absorbance-based G-LISA assays? Question 3: How do I reduce high background readings when using the luminescence-based kits (Cat. # BK121 and BK126)?   Activation Conditions and Cell/Tissue Compatibility Question 4: Will the G-LISA assays work for my specific cell type? Question 5: How can I activate Rho, Rac or Cdc42 GTPases? Question 6: How should I create a cell extract for testing the G-LISA kits? Question 7: Can I use the same lysis buffer to prepare lysates for use in the RhoA, Rac and Cdc42 G-LISA assays? Question 8: Can I use the G-LISA kits to measure GTPase activity in tissue lysates?   3-D/3D cultures and G-LISAs Question 9: Can G-LISA assays be used with cells grown in matrigel or other 3-D growth conditions?   Detection of GTPase Isoforms Question 10: Can I use the G-LISA kits to detect other isoforms of the Rho, Rac and Ral GTPases?   Equipment Requirements Question 11: Which plate shakers are suitable for the G-LISA assays?     Question 1. How can I increase the difference in Rho activation levels between control samples and stimulated samples?   Three different solutions to this problem may exist:   A: If your control cells are supposed to be serum-starved, the lack in difference could be due to inefficient serum starvation, i.e. high basal Rho activation levels. To achieve good serum starvation and inactivation of Rho with most cells, it is important to have had the cells growing in the plates for at least 3 days before the starvation is started. We recommend the following protocol for serum starvation:   T=0 days: Plate cells to 3 to 5% confluency (1 to 3x104 cells/ml). T=3 days: At ~30% confluency, exchange media to 0.5% serum for 24 h T=4 days: At ~50% confluency exchange media to 0% serum for overnight (10 to 16 h) T=5 days: Perform the experiment and freeze the lysates for G-LISA later. T=5 days: Perform protein assay, calculate dilutions necessary and perform G-LISA.   B: You could be looking too late or too early to see the maximum effect on Rho activation. We recommend to always do time curves for stimuli that have unclear effects on Rho activation. For example, LPA is a well-known Rho activator that has Rho-dependent effects on cells and their cytoskeleton for prolonged times. The Rho activation seen by LPA treatment, on the other hand, is very transient. The spike in measurable Rho activity is often gone within 10 min.   C: Too much time may have elapsed between the cell lysis and the addition of the concentration-equalized lysates to the G-LISA wells. This will lead to smaller differences between samples. One way to get around this problem, especially if you are handling large numbers of samples, is to snap-freeze the samples in liquid nitrogen as soon as possible after lysis and clarification. To do this, lyse your cells, clarify the lysates, take off 30 µl of lysate for concentration determination and snap freeze the remaining sample. This way, protein concentrations and dilutions needed can be determined without time pressure. Once all samples are prepared and measured with regards to protein concentration, thaw all samples quickly, dilute as needed and add to the G-LISA wells according to the manual.   Question 2. How can I eliminate high variability between samples when using the absorbance-based G-LISA assays?   Two usual causes:   Not flicking and tapping the plate vigorously enough between washes. Check to make sure that there are no bubbles in the wells. Bubbles will affect the readings.   Question 3. How do I reduce high background readings when using the luminescence based kits (Cat. # BK121 and BK126)?   Users have successfully used a gel scanner to read the plate and achieve lower readings with a 12:1 ratio between positive control and background readings. Alternatively, many luminometers have a dynamic range for sensitivity (gain) and integration time. These parameters should be adjusted and evaluated under control conditions to optimize signal detection. It is also important to note that baseline luminescence readings will vary from machine to machine. If the constitutively-active GTPase control protein samples are 3-5 fold higher than lysis buffer blank, the machine is accurately measuring active GTPase levels.   Question 4. Will the G-LISA assay work for my specific cell type?   Since Rho and Rho signaling pathways are highly conserved between species, the G-LISA kits are likely to work for all mammalian species and many other eukaryotic organisms. We are compiling a list of cell lines and species where G-LISA has been tested. See Table 1 at the bottom of this page. If you have used G-LISA with a cell type or species not listed in the table, we would very much like to hear from you. Email us about your experience.   Table 1. Examples of cell/tissue types compatible with G-LISA assays   Question 5. How can I activate Rho, Rac or Cdc42 GTPases?   We recommend preparing positive control sample lysates that can be used with each G-LISA assay. For activation of the Rho GTPase, we recommend treating serum-starved cells for 10-30 min with 0.25-1 unit/ml of calpeptin (cat# CN01). For activation of the Rac GTPase, treatment of serum-starved cells for 2 min with 10 ng/ml of epidermal growth factor is recommended (cat# CN02). For activation of the Cdc42 GTPase, we recommend treatment of serum-starved cells for 1 min with 100 ng/ml of epidermal growth factor (cat# CN02). Additionally, we recommend omission of the PBS wash before lysing cells that have activated Cdc42 as the PBS wash alone can activate this GTPase. Finally, please see the attached table 2 for a list of common activators accompanied by the relevant citation and additional experimental information.   Table 2. Activators of Rho, Rac and Cdc42   Question 6. How should I create a cell extract for testing and setting up the G-LISA assays?   We recommend preparing two control cell extracts that can be used to set up the G-LISA assay with your cell or tissue type. A simple activated/control state pair of extracts can be made by growing cells to 50% confluency in serum-containing media, washing twice with room temperature PBS, preparing lysate as normal, aliquoting and freezing in liquid nitrogen. Defrost one aliquot and let stand at RT for 1h which will degrade the signal to a low signal and will be the control state. A second defrosted aliquot to be used immediately will be considered “activated”, which most serum grown cells are.   Question 7. Can I use the same lysis buffer to prepare lysates for use in the RhoA, Rac and Cdc42 G-LISA assays?   The Rac and RhoA G-LISA kits use the same lysis buffer.  The Cdc42 G-LISA kit (Cat. # BK127) uses a different lysis buffer.  The buffer components are proprietary, but in general, the lysis buffers contain a buffer, detergents and salts.  The Cdc42 lysis buffer is about 2X more concentrated than the other lysis buffer in regard to salt and detergent concentrations. So you could make the extracts in the Cdc42 lysis buffer and dilute them in the other lysis buffer for the Rac1 and RhoA assays.  As a reminder, be sure to aim for approximately 0.5 - 1 mg/ml protein concentration when performing the lysis.  At higher concentrations, you are likely to have significant loss of signal due to proteolysis, increased phosphatase/kinase activity and increased GAP activity.   Question 8. Can I use the G-LISA kits to measure GTPase activity in tissue lysates?   Yes, tissue lysates can be used instead of cell lysates and the general G-LISA instructions should be followed with the following modification. We recommend isolating the tissue, chopping the tissue into 1 mm chunks and macerating as quickly as possible, perhaps as quickly as 2 min total time. The small GTPase signaling pathway is very quick to change and activated GTPases can be hydrolyzed very quickly. Homogenization can be achieved in the lysis buffer with a micro-pestel and mortar (see Fisher or VWR). Alternatively, 1 mm chunks can be drop frozen in liquid nitrogen for later processing. After homogenization, centrifugation is performed to remove cell debri and the protein concentration is determined. Phosphatase inhibitors are necessary for measuring Rho-GTP in artery samples, but we do not know whether other tissues require these. We recommend the following mixture, a combination of false substrates and inhibitors of serine/threonine phosphatases.   NaF, Final 50 mM, 100 µl of 500 mM stock Na pyrophosphate, Final 20 mM, 100 µl of 200 mM stock p-Nitrophenyl phosphate, Final 1mM, 100 µl of 10 mM stock Microcystin LR, Final 1 µM, 2.5 µl of 400 µM stock (in 10% ethanol) Cytoskeleton Lysis buffer, 700 µl Cytoskeleton protease inhibitors (Cat. # PIC02), 10 µl   There are several things to consider when dealing with potential variation in the experimental tissue samples, e.g., GAP activity, GEF activity, phosphatase activity, physical treatment and time to freezing. To reduce the enzyme activities, you can prepare the lysates as described above. A good test for lysates is to see what happens to them when you incubate at RT for 0, 2, 10 and 20 min. If the signal goes down rapidly, then your extracts need stabilizing. Try to aim for an extract concentration of 0.5 mg/ml because then the enzyme activities will be reduced. High activation states under non-stimulated conditions might be due to the GEF activity or the physical treatment during excision (try a new sharp scalpel and slice rather than chop the tissue). 1 mm3 chunks should be frozen in liquid nitrogen. If these suggestions do not help, then try adding 300 mM NaCl and 0.2% SDS to the lysis buffer as this will reduce the GEF and GAP activities by reducing their interaction with the small G-proteins.   Question 9. Can G-LISA assays be used for cells grown in matrigel or other 3-D growth conditions?   Cells grown in 3-D culture in Matrigel or collagen gels can be assayed using the luminescence-based (BK121 and BK126) kits. The low amounts of cells in 3D cultures makes the conventional pull-down assay very difficult, variable and expensive. Customers have successfully used the G-LISA assay with cells grown in these conditions. Please see Keely et al., 2007 (Methods in Enzymology, v426, p27) and Petroll et al., 2008 (J Cell Physiol, v217, p162) for in-depth descriptions using G-LISA technology and 3-D cultures. An additional modification we suggest is to increase the ratio of extract/binding buffer from the recommended 1:1 (v/v) to 1:3.   Question 10. Can I use the G-LISA kits to detect other isoforms of the Rho, Rac and Ral GTPases?   The RhoA G-LISA kit (Cat. # BK121 and BK124) can be used to detect RhoB or RhoC. The Rac G-LISA (Cat. # BK125 and BK128) can be used to detect Rac2 or Rac3. The RalA G-LISA (Cat. # BK129) can be used to detect RalB. In all cases, the G-LISA wells capture multiple isoforms of the respective GTPase and the specificity of the primary antibody added to the wells determines which activated isoforms are detected and quantified. We recommend evaluating antibodies by western blotting of purified isotype-specific G-proteins and then titrating these verified primary antibodies with the respective G-LISA kit. Please see Hall et al., 2008 (Neoplasia. v10, p797) for a successful example of this modification of G-LISA technology. Recently, a customer has also reported that a RhoB antibody available from Santa Cruz worked quite well with our RhoA G-LISA kit.   Question 11. Which plate shakers are suitable for the G-LISA assays?   At Cytoskeleton, Inc., we use titer plate shakers (model# 4625) from Lab-Line Instruments. These models are also compatible:   Model # RF7854  Digital Microplate Shaker, ML Market Lab,  researchml.com Model # RF7855  Incubating Microplate Shaker, ML Market Lab, researchml.com   G-LISA is a registered trademark of Cytoskeleton, Inc (CO). All rights reserved.