To evaluate Rac1 activity, a Rac1 Pull-down Activation Assay Biochem Kit (Cytoskeleton) was employed. Mouse primary chondrocytes were plated at a density of 1 × 105 cells in 2 × 2 cm stretch chambers. After cyclic tensile strain (0.5 Hz, 10% elongation) for 30 min, proteins were isolated from cells and 500 µg was used in a pull-down assay with p21-activated kinase–Rac1 p21 binding domain beads. The resulting pull-down product was then immunoblotted with Rac1 antibody according to the manufacturer’s instructions. His-tagged Rac1 protein in the kit was used for control. The membranes were incubated with a horseradish peroxidase-conjugated antibody (Promega), and visualized with ECL prime (GE Healthcare) and an AE-6981 Light Capture II (ATTO, Tokyo, Japan). Control cells were seeded onto identical chambers and cultured without cyclic tensile strain. Original images of the assay were shown in Supplementary Fig. 15 .
Rac1 pull down activation assay biochem kit
Manufactured by Cytoskeleton
Sourced in United States
The Rac1 Pull-down Activation Assay Biochem Kit is a laboratory equipment product designed to detect and measure the activation of the Rac1 protein. The kit includes components necessary to perform the pull-down assay, which is a biochemical technique used to isolate and quantify the active, GTP-bound form of Rac1 from cell lysates.
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Lab products found in correlation
Ecl prime, by GE Healthcare (1 mentions)
Restore plus western stripping buffer, by Thermo Fisher Scientific (1 mentions)
Novex ecl, by Thermo Fisher Scientific (1 mentions)
Criterion blotter apparatus, by Bio-Rad (1 mentions)
Gel doc, by Bio-Rad (1 mentions)
Ecl kit, by GE Healthcare (1 mentions)
Anti myc antibody, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Protease inhibitors and phosphatase inhibitor, by Roche (1 mentions)
Accutase, by Merck Group (1 mentions)
Ix71 microscope, by Chroma Technology (1 mentions)
12 protocols using rac1 pull down activation assay biochem kit
1
Rac1 Activity Assessment in Chondrocytes
2
Activated RAC1 Quantification Protocol
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Activated RAC1 was collected using a Rac1 Pull-down Activation Assay Biochem Kit (Cytoskeleton Inc., Denver, CO, USA) according to the manufacturer’s instructions. Assays were conducted on cells harvested on the 4th–7th days after the addition of 4-OHT or DOX. Active RAC1, as well as total RAC1, was quantified by immunoblotting using anti-RAC1 antibody.
3
Western Blot and Rac1 Activation Assay
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Total protein was extracted using cell lysis buffer containing protease and phosphatase inhibitor mixtures. Equivalent protein concentrations were loaded into 4–12% NuPAGE Novex Tris acetate gradient gels (ThermoFisher Scientific, Waltham, MA, USA) for electrophoresis. Proteins were subsequently transferred to a nitrocellulose membrane using a Criterion blotter apparatus (Bio-Rad, Hercules, CA, USA). The membrane was incubated in 5% non-fat dry milk in TBST for 1 h. Immunoblots were incubated overnight with primary antibody in TBST at 4 °C. The next day immunoblots receive 3–5 min washes in TBST before incubation with secondary antibody diluted in TBST for 1 h. Detection of immunoreactive bands was performed using chemiluminescence (Novex ECL, Invitrogen, Waltham, MA, USA). Blots were stripped using Restore Plus Western Stripping Buffer (Thermo Fisher Scientific) for 5–10 min at room temperature and reprobed using another primary antibody. Developed X-ray films were imaged and digitized using a Bio-Rad GelDoc with ImageLab software. Pixel intensities for bands were used for quantification after normalization to loading control bands (α-tubulin). Active Rac1-Pulldown was performed as per manufacturer instructions using Rac1 Pull-Down Activation Assay Biochem Kit (Cytoskeleton Inc., Denver, CO, USA).
4
Measuring Rac1 Activity in Xenopus
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To measure Rac1 activity, we used the Rac1 Pulldown Activation Assay Biochem Kit (BK035, Cytoskeleton, Inc.). DMZ explants expressing myc-tagged xlRac1 were treated with Ionomycin for 3 minutes or 2 hours. To detect Rac1 activity, after Ionomycin or DMSO treatment in DFA medium, the DMZ explants were washed with 1 ml of Steinberg’s solution. The collected explants were lysed in lysis buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 0.5 M NaCl, and 2% Igepal) with a cocktail of protease inhibitors. The supernatant of the lysed sample was collected and used for a pull-down assay with GST-tagged PAK-PBD beads, which bind to active Rac1. The samples were denatured by adding an equivalent volume of 2x SDS sample buffer (0.5 M Tris–HCl pH 6.8, 10% SDS, 50% glycerine, 5% 2-mercaptoethanol). After boiling for 5 min, the samples were subjected to SDS-PAGE and blotted onto PVDF membranes (Bio- Rad). For detection, an anti-myc antibody (#2272 Cell Signaling), HRP-conjugated secondly antibody, and ECL kit (GE Healthcare) were used.
5
Rac1 Activation Assay Protocol
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Cells were lysed in Mg2+ lysis buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10 mM MgCl2, 1 mM EDTA, and 10% glycerol) containing protease inhibitors and phosphatase inhibitors (Roche). The cell lysates were then incubated with GST-PAK-CRIB coupled to glutathione-Sepharose 4 beads using RAC1 pull-down activation assay biochem kit (Cytoskeleton, Inc.) for 1.5 h at 4°C. The beads were washed in lysis buffer, re-suspended in SDS–PAGE sample buffer, and analyzed by Western blotting with anti-Rac1 antibody.
6
Rac1 Activity Determination by Pull-down Assay
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Rac1 activity was tested following the manufacturer’s protocol with Rac1 Pull-down Activation Assay Biochem Kit (BK035, Cytoskeleton). Briefly, lysates were mixed with GST-Pak-PBD beads for 1 h at 4 °C. After washing, the pelleted beads were resuspended in Laemmli sample buffer and subjected to SDS–polyacrylamide gel electrophoresis. GTP-bound Rac1 was detected by western blot analysis using anti-Rac1 antibody. The total amount of Rac1 was detected by immunoblotting of the whole cell lysates.
7
Rac1 Activation Assay in VEGF-Treated Endothelial Cells
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Human endothelial cells were grown to a density of approximately 70% and transfected with a control siRNA or siRNA directed against AKAP12. After 24 hours, cells were detached from the culture dishes using accutase (Merck) and split in a ratio of 1:2 (to keep cells sub‐confluent) and after a further 24 hours were treated with VEGF (50 ng/mL, 30 minutes). Cells were washed with ice‐cold PBS and samples were further processed using the Rac1 Pull‐down Activation Assay Biochem Kit (Cytoskeleton, Denver, USA) according to the manufacturer’s protocol. 1200 µg cell lysate per sample was used to pull down active Rac1.
8
Rac1 Activity Measurement Using Pull-Down Assay
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Rac1 activity was measured using the Rac1 Pull-Down Activation Assay Biochem Kit (Cytoskeleton Inc.). Briefly, HLMVEC and mouse lung tissue were lysed using the cell lysis buffer and the lysate with PAK-PBD beads was incubated at 4 °C on a rotator for 1 h. The PAK-PBD beads were centrifuged at 5000 g and washed with wash buffer. Equal volume of 2x Laemmli sample buffer was added to each tube and boiled for 5 min. Samples were analyzed by SDS-PAGE and Western blot analysis as described above.
9
Rac Activation Assay Protocol
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Rac activation assays were performed according to the manufacturer’s protocol of RAC1 pull-down activation assay biochem kit (Cytoskeleton, United States).
10
Rac1 Activity Measurement using Pull-down Assay
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RAC1 activity was evaluated using Rac1 Pull‐down Activation Assay Biochem Kit (Cytoskeleton). In brief, cell lysis buffer was used to lyse the cells under different treatments (at 24 h after treatment with drug or 48 h after RAC1 transfection), and western blot was performed for quantification of total RAC1 using the collected lysates. The mixture of the sample and 20 μg GST‐PAK PBD beads, which bind to the active GTP‐RAC1 form, was rotated at 4 °C for 1 h. Beads were washed by wash buffer and resuspended by loading buffer. Proteins were separated on 10% SDS/PAGE and transferred to PVDF membranes for western blot. The total and activated RAC1 were detected by western blot using an anti‐RAC1 monoclonal antibody as described by the manufacturer.
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