Rac1 activation assay kit

Manufactured by Abcam

Sourced in United States

The Rac1 activation assay kit is a tool designed to measure the activation state of the Rac1 protein. It provides a quantitative assessment of the amount of active, GTP-bound Rac1 present in a sample.

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Lab products found in correlation

Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Anti stat1, by Cell Signaling Technology (1 mentions) Anti ikkα, by Cell Signaling Technology (1 mentions) Anti p50, by Santa Cruz Biotechnology (1 mentions) Anti akt, by Cell Signaling Technology (1 mentions) Anti osteopontin, by Abcam (1 mentions) Anti blimp 1, by Abcam (1 mentions) Anti p65 c 20, by Santa Cruz Biotechnology (1 mentions) Anti lamin b m 20, by Santa Cruz Biotechnology (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco s modified eagle s, by Thermo Fisher Scientific (1 mentions) Anti rac1 antibody, by Abcam (1 mentions)

5 protocols using rac1 activation assay kit

Immunoblotting and Rac1 Activation Assay

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Cell cytoplasmic and nuclear extracts were obtained by using NE-PER nuclear and cytoplasmic extraction reagents according to the manufacturer’s protocols (ThermoFisher). Immunoblotting was performed as previously described (8 (link)). The blots were probed with anti-IRF7 antibody (EPR4718; abcam), anti-Lamin-B (M-20; Santa Cruz Biotechnology), anti-IKKα (Cell Signaling), anti-AKT (Cell Signaling), anti-Osteopontin (Abcam), anti-p65 (C-20; Santa Cruz Biotechnology), anti-P50 (Santa Cruz Biotechnology), anti-STAT1 (Cell Signaling), anti-IRAK-M (ProSci), and anti-Blimp-1 (Abcam). The activation of IRF7, IKKα/β, AKT, and STAT1 were detected by phospho-specific antibodies against pIRF7 (Ser471/472; D6M2I; Cell Signaling), pIKKα/β (Ser176/180; 16A6; Cell Signaling), pAKT (Ser473; D9E; Cell Signaling), and pSTAT1 (Tyr701; 58D6; Cell Signaling). Representative blots from at least two independent experiments were shown.
Rac1 activation was detected by Rac1 activation assay kit (Abcam). Briefly, the total cell lysates were harvested from stimulated FLpDCs and incubated with PAK1 PBD beads at 4°C for 1 h. Rac1-GTP precipitate and the total lysate controls were analyzed by western blot analysis. Rac1 was detected by a specific mouse monoclonal antibody.

Ko Y.A., Chan Y.H., Liu C.H., Liang J.J., Chuang T.H., Hsueh Y.P., Lin Y.L, & Lin K.I. (2018). Blimp-1-Mediated Pathway Promotes Type I IFN Production in Plasmacytoid Dendritic Cells by Targeting to Interleukin-1 Receptor-Associated Kinase M. Frontiers in Immunology, 9, 1828.

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Rac-GTPase Activation Assay in HEK293T Cells

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Three constructs (V5 empty, V5 wild-type CHN1, and V5 variant p.(Phe213Val) CHN1) were constructed based on the pCDNA3.1 (+) vector and were transiently transfected into the human embryonic kidney (HEK) 293T cells. HEK293T cells were cultured at 37 °C in an incubator with 10% CO₂. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 1% glutamine, and 1% of a double-antibody. After 48 h in culture, a Rac-GTP activation assay was conducted using a Rac1-activation assay kit (Abcam, Cambridge, MA, USA), which was performed according to the manufacturer’s instructions. Briefly, cells were solubilized in lysis buffer. PAK1 PBD agarose beads were used to selectively pull-down active Rac from protein extracts. Subsequently, the precipitated GTP-Rac was detected via Western blotting using a mouse anti-Rac1 specific monoclonal antibody (BD Transduction Laboratories, San Jose, CA). β-actin was also detected—which was used as an internal reference—using β-actin mAB (Abmart, P3002, Arlington, MA, USA) and HRP-conjugated goat Anti-rabbit IgG (CST, 7074, USA). Secondary antibodies and signals were detected with an ECL detection system.

Zhou T.C., Duan W.H., Fu X.L., Zhu Q., Guo L.Y., Zhou Y., Hua Z.J., Li X.J., Yang D.M., Zhang J.Y., Yin J., Zhang X.F., Zhou G.L, & Hu M. (2020). Identification of a novel CHN1 p.(Phe213Val) variant in a large Han Chinese family with congenital Duane retraction syndrome. Scientific Reports, 10, 16225.

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Quantifying Active Rac1 in Brain Lysates

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The activation form of Rac1 (GTP-Rac1) was obtained by a pull-down assay using Rac1 activation Assay kit (Abcam, USA). According to the instructions, brain lysates were incubated with PAK1 PBD bead at 4°C for 1 h. The eluted proteins were then detected by performing Western blotting with anti-Rac1 antibody (Abcam, USA) as described above.

Liu W., Huang J., Doycheva D., Gamdzyk M., Tang J, & Zhang J.H. (2019). RvD1binding with FPR2 attenuates inflammation via Rac1/NOX2 pathway after neonatal hypoxic-ischemic injury in rats. Experimental neurology, 320, 112982.

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Rac1 Activity Assay Protocol

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Rac1 activity was detected using a Rac1 Activation Assay Kit (ab211161, Abcam) according to the product instruction. GST-PBD (p21-binding domain of PAK) was used for the Rac1 activity assays. The CRC cells were transfected with DMTN overexpression, knockdown, or control vector and washed with PBS after 72 h. Then, the cells were lysed in ice-cold Mg²⁺ lysis buffer and centrifuged for 5 min at 13,000× g at 4 °C. Next, 40 μL of supernatant was taken to determine the total Rac1 levels. The remaining supernatants were incubated with GST-PBD on glutathione-sepharose beads and rotated at 4 °C for 2 h. The beads were washed extensively in lysis buffer, and the bound proteins were separated by SDS-PAGE and then immunoblotted with anti-Rac1 antibodies.

Cai L., Gao L., Zhang G., Zeng H., Wu X., Tan X., Qian C, & Chen G. (2022). DJ-1 Alleviates Neuroinflammation and the Related Blood-Spinal Cord Barrier Destruction by Suppressing NLRP3 Inflammasome Activation via SOCS1/Rac1/ROS Pathway in a Rat Model of Traumatic Spinal Cord Injury. Journal of Clinical Medicine, 11(13), 3716.

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Rac1 Activation Assay in Macrophages

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To detect active, GTP-bound Rac1, a pulldown assay using a Rac1 activation assay kit was performed, according to the manufacturer's instructions (Abcam). In brief, human macrophages were treated with 100 ng/ml LPS, 20 ng/ml IFN-g, and carboxylated modified beads for 30 min, and the cells were lysed in a Rac1 lysis buffer containing protease inhibitors and GST-PAK-CRIB, a GST fusion of CRIB of PAK that selectively precipitates the activated form of Rac1. Extracts from unstimulated and nontransfected cells were spiked with Rac1-GDP and Rac1-GTP-g-S to serve as controls for the specificity of the affinity purification. The spikes were incubated at 30°for 15 min with constant agitation, samples centrifuged, and the supernatant harvested for determination of Rac1 activation. Lysate (40 ml) was collected for total Rac1, which was used as an internal loading control for active Rac1. Glutathione-Sepharose beads were added to the remaining sample lysates and incubated for 30 min under constant mixing at 4°C. The beads and precipitated proteins were washed 3 times with ice-cold lysis buffer. The resulting pulldown (active Rac1) and total Rac1 was then immunoblotted with Rac1 antibody, according to the manufacturer's instructions, and immunoblotted proteins were detected using an ECL-based detection system (GE Healthcare).

Gordon P., Okai B., Hoare J.I., Erwig L.P, & Wilson H.M. (2016). SOCS3 is a modulator of human macrophage phagocytosis. Journal of leukocyte biology, 100(4).

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