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 level of activation is measured with luminescence. For a more detailed introduction on G-LISA™ assays and a listing of other available G-LISA™ kits, see our main G-LISA™ page. The Rac1 G-LISA™ Activation Assay (Cat.# BK126) measures the level of GTP-loaded Rac1 protein in cell lysates, this is in contrast to Cat. BK125 which does not distinguish between activated Rac1,2 or 3. For a kit to measure RhoA activation please check webpage BK124.
The Rac1 G-LISA Activation Assay is very sensitive and has excellent accuracy for duplicate samples. See G-LISA™ FAQs tab on our G-LISA™ page for more details.
The kit contains sufficient reagents to perform 96 Rac activation assays. Since the Rac-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from one to multiple assays. The following components are included in the kit:
Serum starved Swiss 3T3 cells were stimulated with the Rac activating compound EGF and Rac activation was measured with the G-LISA method (Fig 1 and 2).
Figure 1. Rac1 activation by EGF measured by G-LISA™ kit BK126. Swiss 3T3 (mouse) cells were serum starved for 24 h and treated with EGF (10ng/ml for 2.5 min) or buffer only (SS). 10 µg of cell lysates were subjected to the G-LISA™ assay. Luminescence measured over 100 milli-second.
Figure 2. Rac activation by EGF measured by G-LISA™. Swiss 3T3 cells were serum starved (SS) for 24 h and treated with EGF (10 ng/ml for 2 min). 60, 30, 15, 7.5, 2.5 µg of cell lysates were subjected to the G-LISA™ assay. Luminescence was measured over 10 milli-seconds. 500 µg of the same lysates were subjected to the traditional PAK pull-down assay (shown in inset, Cat.# BK035).
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at firstname.lastname@example.org
|Phuyal, Santosh et al.
|Mechanical strain stimulates COPII-dependent secretory trafficking via Rac1
|The EMBO Journal
|Nath, Anu S. et al.
|Modulation of the cell membrane lipid milieu by peroxisomal β-oxidation induces Rho1 signaling to trigger inflammatory responses
|Turgu, Busra et al.
|HACE1 blocks HIF1α accumulation under hypoxia in a RAC1 dependent manner
|Garitano-Trojaola, Andoni et al.
|Actin cytoskeleton deregulation confers midostaurin resistance in FLT3-mutant acute myeloid leukemia
|Zhu, Bili et al.
|Lipid oversupply induces CD36 sarcolemmal translocation via dual modulation of PKCζ and TBC1D1: An early event prior to insulin resistance
|Berthenet, Kevin et al.
|Failed Apoptosis Enhances Melanoma Cancer Cell Aggressiveness
|Crawford, Melissa et al.
|Essential Role for Integrin-Linked Kinase in Melanoblast Colonization of the Skin
|Journal of Investigative Dermatology
|Elshaer, Sally L. et al.
|Modulation of the p75 neurotrophin receptor using LM11A-31 prevents diabetes-induced retinal vascular permeability in mice via inhibition of inflammation and the RhoA kinase pathway
|El-Naggar, Amal M. et al.
|HACE1 is a potential tumor suppressor in osteosarcoma
|Cell Death and Disease
|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
|Kang, Jeong Hun et al.
|Protein kinase Cα as a therapeutic target in cancer
|Protein Kinase C: Emerging Roles and Therapeutic Potential
|Kai, Masahiro et al.
|Epigenetic silencing of diacylglycerol kinase gamma in colorectal cancer
|Jeong, Suk Yeong et al.
|Loss of Tpm4.1 leads to disruption of cell-cell adhesions and invasive behavior in breast epithelial cells via increased Rac1 signaling
|Ruggiero, Carmen et al.
|Dosage-dependent regulation of VAV2 expression by steroidogenic factor-1 drives adrenocortical carcinoma cell invasion
|Kim, Jongshin et al.
|YAP/TAZ regulates sprouting angiogenesis and vascular barrier maturation
|Journal of Clinical Investigation
|Thuault, Sylvie et al.
|The RhoE/ROCK/ARHGAP25 signaling pathway controls cell invasion by inhibition of Rac activity
|Molecular Biology of the Cell
|Sylow, Lykke et al.
|Rac1 governs exercise-stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice
|Journal of Physiology
|Sarowar, Tasnuva et al.
|Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2
|Marchesin, Valentina et al.
|ARF6 promotes the formation of Rac1 and WAVE-dependent ventral F-Actin rosettes in breast cancer cells in response to epidermal growth factor
|Reyes-Reyes, E. Merit et al.
|Mechanistic studies of anticancer aptamer AS1411 reveal a novel role for nucleolin in regulating Rac1 activation
|Yan, Yi et al.
|Augmented AMPK activity inhibits cell migration by phosphorylating the novel substrate Pdlim5
|Sylow, Lykke et al.
|Rac1 - a novel regulator of contraction-stimulated glucose uptake in skeletal muscle
|Radeva, Mariya Y. et al.
|PKA compartmentalization via AKAP220 and AKAP12 contributes to endothelial barrier regulation
|Nakajima, Chikako et al.
|The lipoprotein receptor LRP1 modulates sphingosine-1-phosphate signaling and is essential for vascular development
|Kikuchi, K. et al.
|Protein kinase C iota as a therapeutic target in alveolar rhabdomyosarcoma
|Bray, Kristi et al.
|Cdc42 overexpression induces hyperbranching in the developing mammary gland by enhancing cell migration
|Breast Cancer Research
|Sylow, Lykke et al.
|Rac1 signaling is required for insulin-stimulated glucose uptake and is dysregulated in insulin-resistant murine and human skeletal muscle
|Redhu, Naresh Singh et al.
|Thymic stromal lymphopoietin induces migration in human airway smooth muscle cells
|Eggers, Carrie M. et al.
|STE20-related kinase adaptor protein α (STRADα) regulates cell polarity and invasion through PAK1 signaling in LKB1-null cells
|The Journal of biological chemistry
|Sayedyahossein, Samar et al.
|Essential role of integrin-linked kinase in regulation of phagocytosis in keratinocytes
|Vives, Virginie et al.
|The Rac1 exchange factor Dock5 is essential for bone resorption by osteoclasts
|Binker, Marcelo G. et al.
|TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2
|Biochemical and biophysical research communications
|Kigel, Boaz et al.
|Plexin-A4 promotes tumor progression and tumor angiogenesis by enhancement of VEGF and bFGF signaling
|McHenry, Peter R. et al.
|P190B RhoGAP has pro-tumorigenic functions during MMTV-Neu mammary tumorigenesis and metastasis
|Breast Cancer Research
|Johanna, Gutiérrez Vargas et al.
|Rac1 activity changes are associated with neuronal pathology and spatial memory long-term recovery after global cerebral ischemia
|Kamai, Takao et al.
|Increased Rac1 activity and Pak1 overexpression are associated with lymphovascular invasion and lymph node metastasis of upper urinary tract cancer
|Baumer, Y. et al.
|Role of Rac 1 and cAMP in endothelial barrier stabilization and thrombin-induced barrier breakdown
|Journal of Cellular Physiology
|Heckman-Stoddard, Brandy M. et al.
|Haploinsufficiency for p190B RhoGAP inhibits MMTV-Neu tumor progression
|Chastre, Eric et al.
|TRIP6, a novel molecular partner of the MAGI-1 scaffolding molecule, promotes invasiveness
|The FASEB Journal
|Moniz, Sónia et al.
|WNK2 modulates MEK1 activity through the Rho GTPase pathway
|Ramirez, Servio H. et al.
|Activation of Peroxisome Proliferator-Activated Receptor γ (PPARγ) Suppresses Rho GTPases in Human Brain Microvascular Endothelial Cells and Inhibits Adhesion and Transendothelial Migration of HIV-1 Infected Monocytes
|The Journal of Immunology
|Petroll, W. Matthew et al.
|Dynamic assessment of fibroblast mechanical activity during rac-induced cell spreading in 3-D culture
|Journal of Cellular Physiology
|Pontow, Suzanne et al.
|Antiviral Activity of a Rac GEF inhibitor Characterized with a Sensitive HIV/SIV Fusion Assay
Question 1: There is less than a 2-fold difference in signal intensity between my positive control and lysis buffer blank. Why?
Answer 1: To accurately measure luminescence signal intensity between the positive control and buffer blank, please check the instrument settings on the luminometer (see below for suggestions). We also recommend running some “set-up” experiments with just the buffer blank and positive control to determine optimal settings for detecting the positive control signal 3-5 fold higher than the buffer blank. It is also important to remember to use a fresh control protein tube for each run of positive control samples. Do not store and re-use the positive control.
Gain controls the sensitivity of the machine. Most luminometers do not allow manual alteration of gain and use an auto-calibration or limited calibration function. Turn off auto-settings and auto-calibration to use the machine in manual mode. It is important to contact the luminometer manufacturer or consult the user’s manual to determine the best way to alter the machine sensitivity. If gain can be altered, one should read at low, medium and high gains to determine the reading within the linear range of the assay (positive control should be 3-5X higher than blank). Gain range varies with instrument. For example, gain in the Tecan GmbH SpectroFluor Plus ranges from 0 - 150 (where 150 is the highest).
This parameter can be varied on most machines. It is a good idea to set the machine at the lowest integration time (usually 10 – 100 ms). Integration times greater than 200 ms are likely to read out of the linear range of the assay and may require lowering of gain or dilution of primary and/or secondary antibodies.
Most machines give the shaking option. The recommended setting is 5 sec shake, medium orbital speed before read. This option is not essential to the assay.
Any setting that specifies 96 well flat, white will be sufficient.
Luminescence does not require excitation or emission filters so the filter spaces should be left blank. If this is not an option, excitation can be set at any value and emission should be set between 400-500nm, with 430-445 as optimal setting.
Question 2: My arbitrary luminescence units (ALU) or relative luminescence units (RLU) are very different from what is depicted in the manual. Why?
Answer 2: This is very typical as the luminescence units will vary from luminometer to luminometer based on the machine’s sensitivity and instrument settings. The important information to take note of is what the relationship is between buffer blank and positive control luminescence values. The positive control signal should be 3-5 fold greater than the buffer blank luminescence signal. If that is the case, then the G-LISA assay is functioning in the linear range and experimental samples can now be processed.
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