G-LISA Cdc42 Activation Assay Biochem Kit (Colorimetric format)
Product Uses Include
The G-LISA® series of Small G-Protein Activation Assays are ELISA based assays with which you can measure the GTP form of small G-proteins from lysates of cells or tissues and all in less than 3 h. The Cdc42 G-LISA Activation Assay measures the entire level of GTP-loaded Cdc42 protein in cell lysates. The level of activation is measured by reading at OD490nm. For a more detailed introduction on G-LISA assays and a listing of other available G-LISA kits, see our main G-LISA page. For a kit to measure RhoA activation please check webpage BK124, for Rac1 go to BK128 for Rac1,2,3 go to BK125, for RalA go to BK129.
The Cdc42 G-LISA Activation Assay is very sensitive and has excellent accuracy for duplicate samples. See this section on our G-LISA information resource page.
Serum starved Swiss 3T3 cells were stimulated with the Cdc42 activating compound EGF and Cdc42 activation was measured with the G-LISA method (Fig 1 and 2).
Figure 1. Cdc42 activation by EGF measured by the Cdc42 G-LISA Activation Assay. Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (100 ng/ml for 2 min). Cell lysates (8, 17, 35 µg) were subjected to the G-LISA assay. Data was read at 490 nm. Numbers on top the yellow columns indicate the fold increase in signal caused by EGF activation, you will notice the ratio remains the same with different protein loadings indicating good linearity with different protein loadings. 500 µg of the same lysates were subjected to the traditional PAK pull-down assay (Cat.# BK034) with similar results.
Figure 2. Time course of Cdc42 activation using EGF at 10, 50 and 100ng/ml . Swiss 3T3 cells were serum starved (SS) for 16 h at 1% serum and 8 h with 0% serum and treated with EGF (10, 50 and 100 ng/ml for1.5, 3.0, 6.0, 10 and 30 min). Cell lysates subjected to the G-LISA™ assay and OD was read at 490 nm. The “controlled state” serum starved value (0.22) was subtracted from these samples prior to plotting. At 100 ng/ml the total activation was 2.1 fold or 110% over the controlled state at 1.5 min. This type of analysis can be performed in one afternoon.
The kit contains sufficient reagents to perform 96 Cdc42 activation assays. Since the Cdc42-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from 1 to 96 assays. The following components are included in the kit:
Model # 4625 Titer Plate Shaker, Lab-Line Instruments, Barnstead Intl.(average priced)
Model # RF7854 Digital Microplate Shaker, ML Market Lab, researchml.com (economical priced)
Model # RF7855 Incubating Microplate Shaker, ML Market Lab, researchml.com (deluxe model)
Rac1,2,3 G-LISA Activation Assay, colorimetric format (Cat.# BK125)
Rac1 G-LISA Activation Assay, luminescence format (Cat.# BK126)
RhoA G-LISA Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA Activation Assay, luminescence format (Cat.# BK121)
G-LISA is a registered trademark of Cytoskeleton, Inc (CO). All rights reserved.
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|Vallejo, Daniela et al.||Wnt5a modulates dendritic spine dynamics through the regulation of Cofilin via small Rho GTPase activity in hippocampal neurons||Journal of Neurochemistry||2021||ISSN 1471-4159|
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|Lee, Daniel||miR‑769‑5p is associated with prostate cancer recurrence and modulates proliferation and apoptosis of cancer cells||Experimental and Therapeutic Medicine||2021||ISSN 1792--0981|
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|Wilson, Kitchener D. et al.||Endogenous Retrovirus-Derived lncRNA BANCR Promotes Cardiomyocyte Migration in Humans and Non-human Primates||Developmental Cell||2020||ISSN 1878-1551|
|Wu, Huijuan et al.||Progressive Pulmonary Fibrosis Is Caused by Elevated Mechanical Tension on Alveolar Stem Cells||Cell||2020||ISSN 1097-4172|
|Zhang, Xiao et al.||Elevating EGFR-MAPK program by a nonconventional Cdc42 enhances intestinal epithelial survival and regeneration||JCI Insight||2020||ISSN 2379-3708|
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|Rong, Zhouyi et al.||Activation of FAK/Rac1/Cdc42-GTPase signaling ameliorates impaired microglial migration response to Aβ42 in triggering receptor expressed on myeloid cells 2 loss-of-function murine models||FASEB Journal||2020||ISSN 1530-6860|
|Talamás-Lara, Daniel et al.||Entamoeba histolytica and Entamoeba dispar: Morphological and Behavioral Differences Induced by Fibronectin through GTPases Activation and Actin-Binding Proteins||The Journal of eukaryotic microbiology||2020||ISSN 1550--7408|
|Krueger, Irena et al.||Reelin amplifies glycoprotein VI activation and alphaiib beta3 integrin outside-in signaling via PLC Gamma 2 and Rho GTPases||Arteriosclerosis, Thrombosis, and Vascular Biology||2020||ISSN 1524-4636|
|Wu, Xuping et al.||Wnt5a induces ROR1 and ROR2 to activate RhoA in esophageal squamous cell carcinoma cells||Cancer Management and Research||2019||ISSN 1179-1322|
|Zanin, Juan P. et al.||The p75NTR influences cerebellar circuit development and adult behavior via regulation of cell cycle duration of granule cell progenitors||Journal of Neuroscience||2019||ISSN 1529-2401|
|Yan, Ting et al.||Integrin αvβ3-associated DAAM1 is essential for collagen-induced invadopodia extension and cell haptotaxis in breast cancer cells||Journal of Biological Chemistry||2018||ISSN 1083-351X|
|Santhana Kumar, Karthiga et al.||TGF-β Determines the Pro-migratory Potential of bFGF Signaling in Medulloblastoma||Cell Reports||2018||ISSN 2211-1247|
|Veluthakal, Rajakrishnan et al.||Restoration of glucose-stimulated Cdc42-PAK1 activation and insulin secretion by a selective Epac activator in type 2 diabetic human islets||Diabetes||2018||ISSN 1939-327X|
|Mayer, Louisa et al.||Nbeal2 interacts with Dock7, Sec16a, and Vac14||Blood||2018||ISSN 1528-0020|
|Suraneni, Praveen K. et al.||Dynamins 2 and 3 control the migration of human megakaryocytes by regulating CXCR4 surface expression and ITGB1 activity||Blood Advances||2018||ISSN 2473-9537|
|Kim, Jongshin et al.||YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation||Journal of Clinical Investigation||2017||ISSN 1558-8238|
|Ruggiero, Carmen et al.||Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion||Science Signaling||2017||ISSN 1937-9145|
|Gu, Changkyu et al.||Dynamin autonomously regulates podocyte focal adhesion maturation||Journal of the American Society of Nephrology||2017||ISSN 1533-3450|
|Jones, Eleanor L. et al.||Dendritic Cell Migration and Antigen Presentation Are Coordinated by the Opposing Functions of the Tetraspanins CD82 and CD37||The Journal of Immunology||2016||ISSN 0022--1767|
|Sroka, Jolanta et al.||Lamellipodia and membrane blebs drive efficient electrotactic migration of rat walker carcinosarcoma cells WC 256||PLoS ONE||2016||ISSN 1932-6203|
|Herrera-Martínez, Mayra et al.||Antiamoebic activity of Adenophyllum aurantium (L.) strother and its effect on the actin cytoskeleton of Entamoeba histolytica||Frontiers in Pharmacology||2016||ISSN 1663-9812|
|Martins, Rui et al.||Heme drives hemolysis-induced susceptibility to infection via disruption of phagocyte functions||Nature Immunology||2016||ISSN 1529-2916|
|Xiao, Bin et al.||Extracellular translationally controlled tumor protein promotes colorectal cancer invasion and metastasis through Cdc42/JNK/ MMP9 signaling||Oncotarget||2016||ISSN 1949-2553|
|Zhao, Chen Ze et al.||Inhibition of farnesyl pyrophosphate synthase improves pressure overload induced chronic cardiac remodeling||Scientific Reports||2016||ISSN 2045-2322|
|Tien, Sui Chih et al.||The Shp2-induced epithelial disorganization defect is reversed by HDAC6 inhibition independent of Cdc42||Nature Communications||2016||ISSN 2041-1723|
|Breslin, Jerome W. et al.||Involvement of local lamellipodia in endothelial barrier function||PLoS ONE||2015||ISSN 1932-6203|
|Guo, Yuna et al.||A novel pharmacologic activity of ketorolac for therapeutic benefit in ovarian cancer patients||Clinical Cancer Research||2015||ISSN 1557-3265|
|Yan, Yi et al.||Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5||Nature Communications||2015||ISSN 2041-1723|
|Ochoa-Alvarez, Jhon A. et al.||Antibody and lectin target podoplanin to inhibit oral squamous carcinoma cell migration and viability by distinct mechanisms||Oncotarget||2015||ISSN 1949-2553|
|Hanin, Geula et al.||Competing targets of microRNA-608 affect anxiety and hypertension||Human Molecular Genetics||2014||ISSN 1460-2083|
|Rigano, Luciano A. et al.||Listeria monocytogenes antagonizes the human GTPase Cdc42 to promote bacterial spread||Cellular Microbiology||2014||ISSN 1462-5822|
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|Valtcheva, Nadejda et al.||The orphan adhesion G protein-coupled receptor GPR97 regulates migration of lymphatic endothelial cells via the small GTPases RhoA and Cdc42||Journal of Biological Chemistry||2013||ISSN 0021-9258|
|Dubash, Adi D. et al.||The GEF Bcr activates RhoA/MAL signaling to promote keratinocyte differentiation via desmoglein-1||Journal of Cell Biology||2013||ISSN 0021-9525|
|Ramsay, Alan G. et al.||Chronic lymphocytic leukemia cells induce defective LFA-1-directed T-cell motility by altering Rho GTPase signaling that is reversible with lenali******||Blood||2013||ISSN 1528-0020|
|Yao, Honghong et al.||Nonmuscle myosin light-chain kinase mediates microglial migration induced by HIV Tat: involvement of β1 integrins||FASEB journal : official publication of the Federation of American Societies for Experimental Biology||2013||ISSN 1530--6860|
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|Bray, Kristi et al.||Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration||Breast Cancer Research||2013||ISSN 1465-5411|
|Yang, Jian et al.||Cardiac-specific overexpression of farnesyl pyrophosphate synthase induces cardiac hypertrophy and dysfunction in mice||Cardiovascular Research||2013||ISSN 0008-6363|
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Question 1: Can I use the lysis buffer from the Cdc42 G-LISA activation assay kit (Cat. # BK127) to prepare samples for the other G-LISA assays?
Answer 1: The Rac1 and RhoA G-LISAs use the same lysis buffer (Part # GL36). The Cdc42 G-LISA kit (Cat. # BK127) uses a different lysis buffer (Part # GL35). The buffer components are proprietary, but in general, the lysis buffers contain a buffer, detergents and salts. GL35 is about 2X more concentrated than GL36 in regard to salt and detergent concentrations. So you could make the extracts in GL35 and dilute them in GL36 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 2: How many cell culture plates can I process at one time during the lysis step?
Answer 2: We recommend that from the point at you add lysis buffer to the plate on ice to aliquoting and snap-freezing the lysate samples in liquid nitrogen, no more than 10 min are allowed to elapse. After 10 min on ice, we find that GTP bound to GTPases (activated GTPases) undergoes rapid hydrolysis. Rapid processing at 4°C is essential for accurate and reproducible results. The following guidelines are useful for rapid washing of cells.
a. Retrieve culture dish from incubator, immediately aspirate out all of the media and place firmly on ice.
b. Immediately rinse cells with an appropriate volume of ice cold PBS (for Cdc42 activation, skip this step and simply aspirate the media) to remove serum proteins.
c. Aspirate off all residual PBS buffer. This is essential so that the Lysis Buffer is not diluted. Correct aspiration requires that the culture dish is placed at a steep angle on ice for 1 min to allow excess PBS to collect in the vessel for complete removal. As noted, the time period between cell lysis and addition of lysates to the wells is critically important. Take the following precautions:
1. Work quickly.
2. Keeping solutions and lysates embedded in ice so that the temperature is below 4°C. This helps to minimize changes in signal over time.
3. We strongly recommend that cell lysates be immediately frozen after harvest and clarification. A sample of at least 20 μl should be kept on ice for protein concentration measurement. The lysates must be snap frozen in liquid nitrogen and stored at -70°C. Lysates should be stored at -70°C for no longer than 30 days.
4. Thawing of cell lysates prior to use in the G-LISA assay should be in a room temperature water bath, followed by rapid transfer to ice and immediate use in the assay.
If you have any questions concerning this product, please contact our Technical Service department at firstname.lastname@example.org.