Active ras detection kit

Manufactured by Cell Signaling Technology

Sourced in United States, Germany

The Active Ras Detection Kit is a tool for the detection and measurement of active Ras proteins. It utilizes a Ras-binding domain to specifically capture the active, GTP-bound form of Ras. The kit includes the necessary reagents and protocols for this purpose.

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Lab products found in correlation

Rabbit anti phospho erk1 2 p44 42 mapk thr202 tyr204 antibody, by Cell Signaling Technology (2 mentions) Anti p44 42 mapk erk1 2 antibody, by Cell Signaling Technology (2 mentions) Mouse anit ha antibody 26183, by Thermo Fisher Scientific (2 mentions) Nucblue live readyprobe, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Ecl western blotting detection reagent, by GE Healthcare (1 mentions) Nupage gel, by Thermo Fisher Scientific (1 mentions) Synergy h1 plate reader, by Agilent Technologies (1 mentions) Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions) Cell counting kit 8 (cck8), by Dojindo Laboratories (1 mentions) Prism 5, by GraphPad (1 mentions) Victor3 plate reader, by PerkinElmer (1 mentions) Image lab 6, by Bio-Rad (1 mentions) Mini protean tgx stain free precast gel, by Bio-Rad (1 mentions) Molecular imager gel doctm xr, by Bio-Rad (1 mentions) Bcip nbt color development substrate, by Promega (1 mentions) β actin antibody, by Merck Group (1 mentions) Quantity one 4, by Bio-Rad (1 mentions) Western lightning plus, by PerkinElmer (1 mentions)

32 protocols using active ras detection kit

Detecting Activated Ras Mutants

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Active Ras Detection Kit from Cell Signaling (8821s) was used as described in the manuscript of the product. Briefly, cells expressing K-Ras4B^G12D, K-Ras4B^{G12D/K101D/R102E} and K-Ras4B^{G12D/R41E/K42D} were collected and lysed in ice-cold 1X lysis buffer supplemented with protease inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Each lysate was separated into two tubes. One of them was incubated with GDP and the other one was incubated with GTPγS. Then, samples were incubated with GST-Raf1-RBD. Raf1-RBD binds to activated form of Ras. Initial samples from each sample and eluted samples was run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted with anti-c-Myc antibody to detect K-Ras4B^G12D and mutant forms. Activation of K-Ras4B was calculated by dividing band intensity of GTPγS-incubated samples to the band intensity of Ponceau S which is the loading control. Mutant results are normalized to those of K-Ras4B^G12D.

Muratcioglu S., Aydin C., Odabasi E., Ozdemir E.S., Firat-Karalar E.N., Jang H., Tsai C.J., Nussinov R., Kavakli I.H., Gursoy A, & Keskin O. (2020). Oncogenic K-Ras4B Dimerization Enhances Downstream Mitogen-activated Protein Kinase Signaling. Journal of molecular biology, 432(4), 1199-1215.

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Ras Activation Assay Protocol

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GTP-bound Ras was detected in WCLs obtained from TBP-3743 and TBP-3868 cells treated as above with DMSO, 10 μM PLX4720, 50 nM dasatinib or a combination of both PLX4720 and dasatinib for 24 hours. We used the active Ras detection kit (Cell Signaling Technologies, Beverly, MA) and followed the manufacturer's instructions. In brief, GST-bound CRAF (RAF1) Ras binding domain (RBD) was incubated with 500 μg WCLs. Following sample preparation, input whole cell lysates and pull down samples were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed for with antibodies against RAS, pERK(T202/Y204), ERK and tubulin.

Borre P.V., Gunda V., McFadden D.G., Sadow P.M., Varmeh S., Bernasconi M, & Parangi S. (2014). Combined BRAFV600E- and SRC-inhibition induces apoptosis, evokes an immune response and reduces tumor growth in an immunocompetent orthotopic mouse model of anaplastic thyroid cancer. Oncotarget, 5(12), 3996-4010.

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Ras Activity Assay in MPNST Cells

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Ras activities in MPNST cells were examined by the Active Ras Detection Kit (#8821, Cell Signaling Technology) following manufacturer’s instuction. Briefly, the cell lysates were incubated with GST-Raf1-RBD and glutathione resin. After wash and centrifugation, the bound fractions of Ras were dissociate and denatured in SDS sample buffer with DTT, and examined by anti-Ras western blotting. Pre-incubating the lysates with 0.1 mM GTPγS or 1 mM GDP before binding with GST-Raf1-RBD created positive or negative control, respectively. Experiments were repeated twice.

Bai R.Y., Esposito D., Tam A.J., McCormick F., Riggins G.J., Clapp D.W, & Staedtke V. (2019). Feasibility of Using NF1-GRD and AAV for Gene Replacement Therapy in NF1-Associated Tumors. Gene therapy, 26(6), 277-286.

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Ras-GTP Detection Assay

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RAS-GTP levels were detected using an Active Ras detection kit, following the manufacturer instructions (Cell Signaling). Cells were lysed in RIPA lysis buffer (ThermoFisher Scientific). Lysates were denatured in 2× sample buffer at 55 °C for 5 min, resolved on 4–12% NuPAGE gels (Invitrogen) and transferred onto PVDF (polyvinylidene fluoride) membranes. Membranes were blocked in 3% BSA in TBST buffer (TBS with Tween-20) for 1 h at room temperature and probed with primary antibodies (Table S1). Bound antibodies were detected with peroxidase-labeled goat anti-mouse IgG or goat anti-rabbit IgG (R&D Systems) and developed with ECL western blotting detection reagents (GE Healthcare).

Hayashi T., Desmeules P., Smith R.S., Drilon A., Somwar R, & Ladanyi M. (2017). RASA1 and NF1 are preferentially co-mutated and define a distinct genetic subset of smoking-associated non-small cell lung carcinomas sensitive to MEK inhibition.. Clinical cancer research : an official journal of the American Association for Cancer Research, 24(6), 1436-1447.

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Detecting Active RAS in A549 Cells

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To detect active RAS in A549 cells, RAS-GTP pulldown was performed according to the manufacturer's instructions using the Active Ras Detection Kit (Cell Signaling Technology, #8821). In brief, one confluent 10 cm dish of cells was rinsed with ice-cold PBS and lysed in 0.5 ml ice-cold lysis buffer supplemented with 1 mM PMSF. Cell lysates were cleared by centrifugation (16,000 g for 15 min at 4°C) and protein concentration was determined by Bradford assay. Cleared lysates were added to the pre-washed spin cup which contains 100 μl of the 50% resin slurry and 80 μg of GST-Raf1-RBD and incubated at 4°C for 1 h on a rotor. The resin was washed three times with Wash Buffer and the proteins bound to the resin were eluted with 50 μl of the sample buffer supplemented with 200 mM DTT. Samples were denatured at 95°C for 5 min and were subjected to western blot analysis.

Sieber B., Lu F., Stribbling S.M., Grieve A.G., Ryan A.J, & Freeman M. (2022). iRhom2 regulates ERBB signalling to promote KRAS-driven tumour growth of lung cancer cells. Journal of Cell Science, 135(17), jcs259949.

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Quantifying TSLP-Induced RAS Activation

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Cells were left uninduced or induced with human TSLP at 37 °C. Whenever indicated, DMSO or inhibitors were added for 3 h before TSLP-induction. Cells were lysed on ice at 1000 cells/µL lysis buffer according to the manufacturer’s protocol of the active RAS detection kit (Cat.#8821; Cell Signaling Technology, Danvers, US). Total protein concentrations of samples were measured using a BCA protein-assay kit (Cat.#23225; ThermoFisher Scientific, Waltham, US). 50 µg total protein was loaded per column of the active RAS detection kit for Western blot (WB). In the RAS activation assay kit for ELISA (Cat.#17-497; EMD Millipore, Burlington, US), 12 µg total protein was used at 100 ng/µL and the RAS-GTP pull-down was measured using a Synergy H1 plate reader (BioTek, Winooski, US) in luminescent mode.
For methods on proximity ligation assay (PLA), principal-component-analysis (PCA), statistical analysis, as well as lists of antibodies/chemicals, and standard protocols for the sequencing, SDS-PAGE/WB, phospho-protein antibody-microarray, and transduction please see “Supplementary Methods”.

Koschut D., Ray D., Li Z., Giarin E., Groet J., Alić I., Kham S.K., Chng W.J., Ariffin H., Weinstock D.M., Yeoh A.E., Basso G, & Nižetić D. (2020). RAS-protein activation but not mutation status is an outcome predictor and unifying therapeutic target for high-risk acute lymphoblastic leukemia. Oncogene, 40(4), 746-762.

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Quantifying Active RAS GTPase

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Cells were seeded in 10-cm dishes. The following day, the 70–80% confluent cells were collected, and GTP-bound RAS was quantified using active RAS detection kit (# 8821) from Cell Signaling according to manufacturer instructions.

Wang J., Pollard K., Allen A.N., Tomar T., Pijnenburg D., Yao Z., Rodriguez F.J, & Pratilas C.A. (2020). Combined inhibition of SHP2 and MEK is effective in models of NF1-deficient malignant peripheral nerve sheath tumors. Cancer research, 80(23), 5367-5379.

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RAS Activation Assay Protocol

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RAF1-RBD immunoprecipitation was performed as previously described [69 ]. Cells were seeded in 10 cm dishes. The following day, the 70–80% confluent cells were collected, and GTP-bound RAS was isolated using the active RAS detection kit (# 8821) from Cell Signaling according to manufacturer instructions. All experiments shown were replicated at least twice.

Odeniyide P., Yohe M.E., Pollard K., Vaseva A.V., Calizo A., Zhang L., Rodriguez F.J., Gross J.M., Allen A.N., Wan X., Somwar R., Schreck K.C., Kessler L., Wang J, & Pratilas C.A. (2022). Targeting farnesylation as a novel therapeutic approach in HRAS-mutant rhabdomyosarcoma. Oncogene, 41(21), 2973-2983.

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Quantifying GTP-Bound RAS in Cells

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Cells were seeded in 10-cm dishes. The following day, the 70% to 80% confluent cells were collected, and GTP-bound RAS was quantified using active RAS detection kit (# 8821) from Cell Signaling Technology according to the manufacturer’s instructions.

Wang J., Calizo A., Zhang L., Pino J.C., Lyu Y., Pollard K., Zhang X., Larsson A.T., Conniff E., Llosa N., Wood D.K., Largaespada D.A., Moody S.E., Gosline S.J., Hirbe A.C, & Pratilas C.A. (2023). CDK4/6 inhibition enhances SHP2 inhibitor efficacy and is dependent upon restoration of RB function in malignant peripheral nerve sheath tumors. bioRxiv.

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Antibody Validation for MAPK Signaling

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The rabbit anti-phospho-Erk1/2 (p44/42 MAPK) (Thr202/Tyr204) antibody (#9101, Lot 23) and anti-Erk1/2 (p44/42 MAPK) antibody (#9102, Lot 27) were purchased from Cell Signaling Technologies. Rabbit anti-NF1 antibody (A300–140A, Lot 3) was purchased from Bethyl laboratories, mouse anit-HA antibody (26183, Lot RJ241582) was purchased from Invitrogen and anti-βActin (C-11, SC-1615HRP, Lot G3015) HRP antibody was purchased from Santa Cruz Biotech. Active Ras Detection Kit (#8821, antibody Lot 7) was purchased from Cell Signaling Technology. NucBlue Live Ready Probes was purchased from Invitrogen.

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