Product Uses Include
The Ras switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state. Understanding the mechanisms that regulate activation / inactivation of Ras-like GTPases is of obvious biological significance and is a subject of intense investigation. The fact that Ras family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors Ras protein activation. The assay uses the Ras-binding domain (RBD) of the Ras effector kinase Raf1. The Raf-RBD domain has been shown to bind specifically to the GTP-bound form of Ras proteins. The fact that the Raf-RBD has a high affinity for GTP-Ras and that its binding results in a significantly reduced intrinsic and catalytic rate of hydrolysis of Ras make it an ideal tool for affinity purification of GTP-Ras from cell lysates. The Raf-RBD is in the form of a GST fusion protein, which allows one to "pull-down" the Raf-RBD/GTP-Ras complex with glutathione affinity beads. The assay therefore provides a simple means of quantitating Ras activation in cells. The amount of activated Ras is determined by a western blot using a Ras specific antibody.
The kit contains enough materials for 50 assays, depending on assay setup, and includes reagents for positive and negative controls. The following components are included:
Figure 1. The brightly colored glutathione agarose beads in kit BK008 makes the kit easy to use..
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
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)
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
|Kirolos, Sara A. et al.||A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent||Molecular biology of the cell||2022||ISSN 1939-4586|
|Jeon, S M et al.||Blockade of PD-L1/PD-1 signaling promotes osteo-/odontogenic differentiation through Ras activation||International Journal of …||2022||Article Link|
|Ablain, Julien et al.||SPRED1 deletion confers resistance to MAPK inhibition in melanoma||The Journal of experimental medicine||2021||ISSN 1540-9538|
|Wiley, Christopher D. et al.||Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis||Cell Metabolism||2021||ISSN 1932-7420|
|Tulpule, Asmin et al.||Kinase-mediated RAS signaling via membraneless cytoplasmic protein granules||Cell||2021||ISSN 1097-4172|
|Yan, Wupeng et al.||Structural Insights into the SPRED1-Neurofibromin-KRAS Complex and Disruption of SPRED1-Neurofibromin Interaction by Oncogenic EGFR||Cell Reports||2020||ISSN 2211-1247|
|Hood, Fiona E. et al.||Isoform-specific Ras signaling is growth factor dependent||Molecular Biology of the Cell||2019||ISSN 1939-4586|
|Cai, Diana et al.||Identification and characterization of oncogenic SOS1 mutations in lung adenocarcinoma||Molecular Cancer Research||2019||ISSN 1557-3125|
|Cocco, Emiliano et al.||Resistance to TRK inhibition mediated by convergent MAPK pathway activation||Nature Medicine||2019||ISSN 1546-170X|
|Rijal, Ramesh et al.||An endogenous chemorepellent directs cell movement by inhibiting pseudopods at one side of cells||Molecular Biology of the Cell||2019||ISSN 1939-4586|
|Pilling, Darrell et al.||Different Isoforms of the Neuronal Guidance Molecule Slit2 Directly Cause Chemoattraction or Chemorepulsion of Human Neutrophils||The Journal of Immunology||2019||ISSN 0022--1767|
|Brandt, Raphael et al.||Cell type-dependent differential activation of ERK by oncogenic KRAS in colon cancer and intestinal epithelium||Nature Communications||2019||ISSN 2041-1723|
|Dardaei, Leila et al.||SHP2 inhibition restores sensitivity in ALK-rearranged non-small-cell lung cancer resistant to ALK inhibitors||Nature Medicine||2018||ISSN 1546-170X|
|Saikia, Minakshi et al.||Heteronemin, a marine natural product, sensitizes acute myeloid leukemia cells towards cytarabine chemotherapy by regulating farnesylation of Ras||Oncotarget||2018||ISSN 1949-2553|
|Smolkin, Tatyana et al.||Complexes of plexin-A4 and plexin-D1 convey semaphorin-3C signals to induce cytoskeletal collapse in the absence of neuropilins||Journal of Cell Science||2018||ISSN 1477-9137|
|Whiteside, Theresa L.||Therapeutic targeting of oncogenic KRAS in pancreatic cancer by engineered exosomes||Translational Cancer Research||2017||ISSN 2219-6803|
|Malchers, Florian et al.||Mechanisms of primary drug resistance in FGFR1-amplified lung cancer||Clinical Cancer Research||2017||ISSN 1557-3265|
|Lu, Xinyuan et al.||MET exon 14 mutation encodes an actionable therapeutic target in lung adenocarcinoma||Cancer Research||2017||ISSN 1538-7445|
|Walton, Josephine B. et al.||CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1, Pten and Nf1, and correlation with platinum sensitivity||Scientific Reports||2017||ISSN 2045-2322|
|Ilinskaya, Olga N. et al.||Direct inhibition of oncogenic KRAS by Bacillus pumilus ribonuclease (binase)||Biochimica et Biophysica Acta - Molecular Cell Research||2016||ISSN 1879-2596|
|Hrustanovic, Gorjan et al.||RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK-positive lung cancer||Nature Medicine||2015||ISSN 1546-170X|
|Larribere, Lionel et al.||NF1 loss induces senescence during human melanocyte differentiation in an iPSC-based model||Pigment Cell and Melanoma Research||2015||ISSN 1755-148X|
|Du, Chang Qing et al.||Inhibition of farnesyl pyrophosphate synthase prevents norepinephrine- induced fibrotic responses in vascular smooth muscle cells from spontaneously hypertensive rats||Hypertension Research||2014||ISSN 0916-9636|
|Yan, Chao et al.||Discovery and characterization of small molecules that target the GTPase Ral||2014||ISSN 1476--4687|
|Ciaglia, Elena et al.||N6-isopentenyladenosine, an endogenous isoprenoid end product, directly affects cytotoxic and regulatory functions of human NK cells through FDPS modulation||Journal of Leukocyte Biology||2013||ISSN 1938--3673|
|Stoppa, Giovanna et al.||Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells||Apoptosis : an international journal on programmed cell death||2012||ISSN 1573--675X|
|Kern, F. et al.||Essential, non-redundant roles of B-Raf and Raf-1 in Ras-driven skin tumorigenesis||Oncogene 2013 32:19||2012||ISSN 1476--5594|
|Kowluru, Renu A.||Role of Matrix Metalloproteinase-9 in the Development of Diabetic Retinopathy and Its Regulation by H-Ras||Investigative Ophthalmology & Visual Science||2010||ISSN 0146-0404|
|Jiang, Xiao Sheng et al.||Activation of Rho GTPases in Smith-Lemli-Opitz syndrome: pathophysiological and clinical implications||Human molecular genetics||2010||ISSN 1460--2083|
|Rose, A. et al.||Stimulatory effects of the multi-kinase inhibitor sorafenib on human bladder cancer cells||British Journal of Pharmacology||2010||ISSN 0007-1188|
|Lito, Piro et al.||Evidence that sprouty 2 is necessary for sarcoma formation by H-Ras oncogene-transformed human fibroblasts||The Journal of biological chemistry||2008||ISSN 0021--9258|
|Wasylyk, Christine et al.||Inhibition of the Ras-Net (Elk-3) pathway by a novel pyrazole that affects microtubules||Cancer Research||2008||ISSN 0008-5472|
|Wang, J. X. et al.||Investigation of the immunosuppressive activity of artemether on T-cell activation and proliferation||British Journal of Pharmacology||2007||ISSN 0007-1188|
Question 1: I have high background and/or multiple bands on my western blot. How can I fix this?
Answer 1: There are multiple causes of high background and/or multiple bands. Some suggestions to improve background signal include:
Question 2: How much of the beads should I use for my pull-down experiments?
Answer 2: Raf-RBD beads (Cat. # RF02) will bind to Ras-GDP with a much lower affinity than Ras-GTP. If too many Raf-RBD beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) Ras. The result of this will be an underestimation of Ras activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Ras activation or inactivation assay. Once optimal conditions have been established, bead titrations should no longer be necessary. We recommend 20, 40 and 60 μl (66, 132 and 198 µg) bead titrations.
Question 3: How can I test whether the beads are working properly?
Answer 3: A standard biological assay for Raf-RBD beads consists of a Ras protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP. Here are guidelines to follow (see Cat. # RF02 or BK008 datasheets for more details):
Positive Cellular Protein Control:
Total cell lysate (200 – 500 µg) should be loaded with GTPγS as a positive control for the pull-down assay. The following reaction details how to load endogenous Ras with the nonhydrolysable GTP analog (GTPγS). This is an excellent substrate for Raf-RBD beads and should result in a strong positive signal in a pull-down assay.
a) Perform GTP loading on 200 – 500 μg of cell lysate that is at a protein concentration between 0.4 – 2.0 mg/ml by adding 1/10th volume of Loading Buffer.
b) Immediately add 1/100th volume of GTPγS (200 μM final concentration). Under these conditions, 5 - 10% of the Ras protein will load with non-hydrolysable GTPγS and will be “pulled-down” with the Raf-RBD beads in the assay.
c) Incubate the control sample at 37°C for 30 minutes with gentle rotation.
d) Stop the reaction by transferring the tube to 4°C and adding 1/10th volume of STOP Buffer.
e) Use this sample in a pull-down assay immediately.
Negative Cellular Protein Control:
This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTPγS. Loading endogenous Ras with GDP will inactivate Ras and this will bind very poorly to Raf-RBD beads.
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