Cell culture dishes were coated with fibronectin at 5 μg/ml or Fbln7-C at 20 μg/ml overnight at 4°C. HUVECs were plated and allowed to attach for 2 hours. Rac1 activity was assayed using the Rac1 Activation Assay Kit from Cytoskeleton Inc. (USA). Cell lysates were incubated with PAK-PBD agarose beads at 4°C for 1 hour. After washing, the proteins attached to the beads were solubilized by boiling in a LDS-sample buffer with β-mercaptoethanol. SDS-polyacrylamide gel electrophoreses (PAGE) was performed, and the proteins were detected by immunoblotting using a Rac1 antibody (Merck, IRL). RhoA activity was assayed using the RhoA Activation Assay Kit from Cytoskeleton. Cell lysates were incubated with rhotekin RBD agarose beads at 4°C for 1 hour. After washing, the proteins attached to the beads were solubilized by boiling in a LDS-sample buffer with β-mercaptoethanol. SDS-PAGEs were performed, and the proteins were detected by immunoblotting using a RhoA antibody (Abcam, USA).
Rac1 activation assay kit
Manufactured by Cytoskeleton
Sourced in United States
The Rac1 activation assay kit is a laboratory equipment designed to measure the activation state of Rac1, a small GTPase protein. The kit provides the necessary components and protocols to perform the assay, which allows for the quantification of the active, GTP-bound form of Rac1 in cellular samples.
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Lab products found in correlation
Rhoa activation assay kit, by Cytoskeleton (1 mentions)
Rac1 antibody, by Merck Group (1 mentions)
L nna, by Merck Group (1 mentions)
Mdl 12 330a, by Merck Group (1 mentions)
Esi 09, by Biolog (1 mentions)
Pierce ecl solution, by Thermo Fisher Scientific (1 mentions)
Ec basal medium plus supplement pack, by PromoCell (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Leica sp8, by Leica camera (1 mentions)
Ab237734, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Elisa kit, by MyBioSource (1 mentions)
9 protocols using rac1 activation assay kit
1
Rac1 and RhoA Activity Assay
2
Endothelial Cell Barrier Permeability Assay
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HRP‐conjugated anti‐mouse IgG, and rabbit IgG antibodies were purchased from Amersham Biosciences (Heidelberg, Germany); human thrombin from Behring (Marburg, Germany); anti VE‐cadherin (clone TEA 1, mouse IgG) from Beckman Coulter (Krefeld, Germany); ESI‐09 from Biolog (Bremen, Germany); benzonase, forskolin, myristoylated PKI, and Y‐27632, from Calbiochem (Darmstadt, Germany); anti‐phospho‐MLC and anti‐GAPDH antibodies from Cell signaling (Danvers, MA); Rac1 activation assay kit from cytoskeleton (Denver); Pierce® ECL solution from Fischer Scientific (Niederlassung Nidderau, Germany); Alexa‐Flour labelled anti‐mouse IgG and anti‐rabbit IgG antibodies from Invitrogen (Karlsruhe, Germany); anti‐Rac1‐GTP antibody from NewEast Biosciences (King of Prussia, PA); EC basal medium plus supplement pack from PromoCell (Heidelberg, Germany); Complete® protease inhibitor cocktail from Roche (Mannheim, Germany); 3‐isobutyl‐1‐methylxanthine (IBMX), Phalloidin‐TRITC, L‐NAME, L‐NNA, MDL‐12 330A, and anti‐vinculin (clone hVIN‐1, mouse IgG) from Sigma (Steinheim, Germany); anti‐phospho‐MYPT1 (Thr850) from Merck Millipore (Schwalbach, Germany), and Transwell polycarbonate membrane filters (24‐mm round) were from Greiner Bio‐One (Frickenhausen, Germany). All other chemicals were of the best available quality, usually analytical grade.
3
Rac1 Activation Assay Protocol
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The Rac1-GTP pulldown assay was performed following manufacturer instructions in the Rac1 activation assay kit (Cytoskeleton, Denver, CO, USA).
4
Docking and Validation of Rac1 and RanGDP Inhibitors
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Computational docking of Pitstop‐2 and RVD‐127 to the crystal structure of RanGDP (PDB code 3GJ0) was performed using AutoDockVina53 software to determine the putative binding sites of Pitstop‐2 and RVD‐127. Prior to docking the inhibitors GDP molecule was removed from the structure. For docking the small molecules to Rac1, the crystal structure of Rac1‐GDP complexed with Arfaptin (PDB Code 1I4D) was used due to the lack of a crystal structure of Rac1 bound to GDP. The Rac1‐GDP model was predicted with help of the modeling web portal Phyre2.54 Docking results were analyzed using AutoDockTools 1.5.655 and the images were prepared using VMD 1.9.2.56 For the in vitro binding of RVD‐127 to recombinant GTPases, Rac1 (Cytoskeleton, Inc., Denver, USA) or Ran (generous gift of Ariberto Fassati) were immobilized on Ni‐NTA affinity beads (Qiagen, Hilden, Germany) and incubated in 100 μM of RVD‐127 in HEPES‐buffered solution (90 mM KCl, 10 mM NaCl, 2 mM MgCl2, 10 mM HEPES, pH 7.4). Confocal imaging of the beads was performed using Leica SP8 (Leica, Wetzlar, Germany). In vitro inhibition of the Rac1 binding to its downstream effector was performed using the Rac1 activation assay kit (Cytoskeleton) per manufacturer's instructions.
5
Evaluating Podocyte Rac1 Activity
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Rac1 activity was evaluated using a Rac1 activation assay. Differentiated podocytes were treated with 3 ng/ml of ADR for 6 h to induce cell damage. Rac1 activity of cultured podocytes in the presence or absence of ADR was assessed using a Rac1 activation assay kit (Cytoskeleton Inc., Denver, CO). Cell lysate (500 mg protein) was prepared and mixed with PAK1-PBD-immobilised beads according to the manufacturer’s protocol. The mixture was incubated at 4 °C for 1 h and centrifuged at 15,000 × g for 3 min. The beads were washed once with washing buffer and centrifuged again. The beads were then treated with SDS-PAGE sample buffer and solubilised protein was subjected to WB analysis for bead-bound (activated) Rac1.
6
Protein Extraction and Western Blot Analysis
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Total protein was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). After quantification, an equal amount of protein was run on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Billerica, MA, United States). After blocking with 5% BSA, the membrane was probed with the indicated antibodies. Primary antibodies used in this study were as follows: C1GALT1 (Abcam, Cambridge, United Kingdom, ab237734), RAC1 (ab155938), and GADPH (ab9485). Reactive bands were visualized using an ECL system (Pierce, Rockford, IL, United States). RAC1 activity was determined using the Rac1 activation assay kit (Cytoskeleton, Denver, CO, United States). GTP-RAC1 was detected by Western blot using an anti-RAC1 antibody.
7
Rac-1 Activation and Plasma Biomarkers Assay
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The activity of Rac‐1 was measured using the Rac‐1 Activation Assay Kit (Cytoskeleton, Inc., Denver, CO, USA), a quantitative ELISA assay that recognizes the active GTP‐bound form of Rac‐1. Plasma levels of uPA, (s)uPAR, Plg and MMPs were measured using commercially available ELISA kits (MyBioSource, San Diego, CA, USA for uPA; Lifescience Market, Hong Kong for (s)uPAR; Abcam for Plg and MMP‐2; LSBio, Seattle, WA, USA for MMP‐9).
8
Rac1 Activation Assay Protocol
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Rac1 activity assays were generated according to the Rac1 activation assay kit (BK035, Cytoskeleton, Denver, CO, USA). In brief, neutrophils were stimulated with 10 μM of fMLP for the indicated times, and then were disrupted with an ice-cold lysis buffer containing a protease inhibitor cocktail. After repeated freezing and thawing, some of the lysates were taken, stored, and used to detect the total Rac1. The remaining cell lysates were incubated with PAK-PBD beads to detect active Rac1. Samples were analyzed by SDS-PAGE and immunoblotting using the anti-Rac1 antibody.
9
Rac1 Activation Assay and Inhibition
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The Rac1 Activation Assay Kit was obtained from Cytoskeleton Inc. (Denver, CO), the Rho Pull-Down and Detection Kit from Thermo Scientific (Rockford, IL), the Rac1 inhibiter (NSC 23766) from Santa Cruz Biotechnology Inc. (San Diego, CA), and the DOCA slowrelease pellets from Innovative Research of America (Sarasota, FL). Antibody sources and dilutions are shown in Table 3. Unless specified, all other reagents were purchased from Sigma-Aldrich Canada Co. (Oakville, ON, Canada).
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