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Rho assay reagent

Manufactured by Merck Group
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Rho assay reagent is a laboratory product manufactured by Merck Group. It is used to measure the activity of the Rho protein, which is a key regulator of cell cytoskeleton and signaling pathways. The reagent enables the quantification of Rho protein levels or activation state in cell-based assays.

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Lab products found in correlation

Rac cdc42 assay reagent, by Merck Group (3 mentions) Laemmli reducing sample buffer, by Bio-Rad (2 mentions) Gtpγs, by Merck Group (2 mentions) Rho activation assay kit, by Merck Group (2 mentions) Mg2 lysis wash buffer mlb, by Merck Group (2 mentions) Anti β actin clone ac 74, by Merck Group (1 mentions) Alliance mini wl2m system, by Uvitec (1 mentions) Anti pdcd10, by Merck Group (1 mentions) Anti cyclin d1 sc 8396, by Santa Cruz Biotechnology (1 mentions) Anti phospho mypt1, by Cell Signaling Technology (1 mentions) Anti phospho mlc2, by Cell Signaling Technology (1 mentions) Anti mlc2, by Cell Signaling Technology (1 mentions) Anti parp, by Cell Signaling Technology (1 mentions) Calf intestinal alkaline phosphatase cip, by New England Biolabs (1 mentions) Rac1 cdc42 activation assay kit, by Merck Group (1 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions) Bca assay, by Thermo Fisher Scientific (1 mentions) Mg2 lysis wash buffer, by Merck Group (1 mentions) Ethylene glycol tetraacetic acid (egta), by Nacalai Tesque (1 mentions) Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)

12 protocols using rho assay reagent

1

Rho Activation Assay in MDA-MB-231 Cells

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Rho activation assays were performed using the Rho Activation Assay Kit (Millipore Sigma #17–924). MDA-MB-231 cells (WT and usp9x KO cells) were grown until confluent, washed in ice-cold 1XPBS, lysed in ice-cold 1X Mg2+ Lysis/Wash buffer (MLB) (Millipore Sigma #20–168), scraped and collected in microfuge tubes on ice, and incubated at 4°C for 15 minutes with agitation. Lysates were then cleared of cellular debris by centrifugation at 14,000×g for 5 minutes at 4°C. Clarified lysate was then bound to Rho Assay Reagent (Millipore Sigma #14–383) at 4°C for 45 minutes with gentle agitation. Agarose beads were then washed 3 times with 1X MLB and proteins were eluted in 2× Laemmli reducing sample buffer (BioRad #1610737) by boiling at 95°C for 5 minutes. Samples were fractionated by SDS-PAGE and analyzed by blotting using the Anti-Rho (−A, −B, −C) clone 55 antibody (Millipore Sigma #05–778).
GTPγS/GDP loading for positive and negative controls were prepared as described above with an additional 30 minute incubation at 30°C with agitation of cell lysate in the presence of 10mM EDTA and either 100uM GTPγS (Millipore Sigma #20–176) or 1mM GDP (Millipore Sigma #20–177). This step was performed prior to binding the Rho Assay Reagent.
Nielsen C.P., Jernigan K.K., Diggins N.L., Webb D.J, & MacGurn J.A. (2019). USP9X Deubiquitylates DVL2 to Regulate WNT Pathway Specification. Cell reports, 28(4), 1074-1089.e5.
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2

Rho Activation Assay in MDA-MB-231 Cells

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Rho activation assays were performed using the Rho Activation Assay Kit (Millipore Sigma #17–924). MDA-MB-231 cells (WT and usp9x KO cells) were grown until confluent, washed in ice-cold 1XPBS, lysed in ice-cold 1X Mg2+ Lysis/Wash buffer (MLB) (Millipore Sigma #20–168), scraped and collected in microfuge tubes on ice, and incubated at 4°C for 15 minutes with agitation. Lysates were then cleared of cellular debris by centrifugation at 14,000×g for 5 minutes at 4°C. Clarified lysate was then bound to Rho Assay Reagent (Millipore Sigma #14–383) at 4°C for 45 minutes with gentle agitation. Agarose beads were then washed 3 times with 1X MLB and proteins were eluted in 2× Laemmli reducing sample buffer (BioRad #1610737) by boiling at 95°C for 5 minutes. Samples were fractionated by SDS-PAGE and analyzed by blotting using the Anti-Rho (−A, −B, −C) clone 55 antibody (Millipore Sigma #05–778).
GTPγS/GDP loading for positive and negative controls were prepared as described above with an additional 30 minute incubation at 30°C with agitation of cell lysate in the presence of 10mM EDTA and either 100uM GTPγS (Millipore Sigma #20–176) or 1mM GDP (Millipore Sigma #20–177). This step was performed prior to binding the Rho Assay Reagent.
Nielsen C.P., Jernigan K.K., Diggins N.L., Webb D.J, & MacGurn J.A. (2019). USP9X Deubiquitylates DVL2 to Regulate WNT Pathway Specification. Cell reports, 28(4), 1074-1089.e5.
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3

Western Blot Analysis of Cellular Signaling

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Whole cell extracts were prepared and transferred to membranes with standard methods as described before [15] (link). Membranes were incubated overnight at 4°C with the following antibodies: anti-PDCD10 (#SAB1305161), anti-β-Actin (clone AC-74, #A2228) from Sigma-Aldrich; anti-phospho cofilin (sc-12912), anti-cofilin (sc-33779) and anti- Cyclin D1 (sc-8396) from Santa Cruz (Dallas, TX, USA); anti-phospho-Mypt1 (#5163), anti-Mypt1 (#2634), anti-phospho-MLC2 (#3671), anti-MLC2 (#8505) and anti-PARP (#9542) from Cell Signaling Technology (Danvers, MA, USA); anti-cleaved Caspase 3 (2305-PC-100) from Trevigen (Gaithersburg, MD, USA). GTP-bound Rho was assayed with Rho assay reagent according to manifacturer instructions (#17-294; Millipore, Burlington, MA, USA). Chemio-luminescence was detected with Alliance Mini WL2M system (Uvitec, Cambridge, UK).
De Marco C., Zoppoli P., Rinaldo N., Morganella S., Morello M., Zuccalà V., Carriero M.V., Malanga D., Chirillo R., Bruni P., Malzoni C., Di Vizio D., Venturella R., Zullo F., Rizzuto A., Ceccarelli M., Ciliberto G, & Viglietto G. (2021). Genome-wide analysis of copy number alterations led to the characterisation of PDCD10 as oncogene in ovarian cancer. Translational Oncology, 14(3), 101013.
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4

Quantifying RhoGTPases Activity

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RhoGTPases activity was assessed using a Rho assay reagent (Millipore, 14-383) and Rac/Cdc42 assay reagent (Millipore, 14-325) as indicated by the manufacturers protocol. Briefly, serum-starved cells were stimulated with EphB2-Fc and then lysed using MLB lysis buffer in the presence of protease inhibitors for 15 min. Rhotekin-agarose or PAK1-agarose was added to cell lysates at a 1:1 volume ratio and incubated at 4°C for 1 hour. After brief centrifugation, the bead pellets were washed with MLB buffer. Laemmli sample buffer was added to the samples and separated by 15% SDS-PAGE, and immunoblotted with anti-RhoA, anti-Rac1 or anti-Cdc42 antibody.
Cho H.J., Hwang Y.S., Yoon J., Lee M., Lee H.G, & Daar I.O. (2017). EphrinB1 promotes cancer cell migration and invasion through the interaction with RhoGDI1. Oncogene, 37(7), 861-872.
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5

Quantifying RhoGTPases Activity

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RhoGTPases activity was assessed using a Rho assay reagent (Millipore, 14-383) and Rac/Cdc42 assay reagent (Millipore, 14-325) as indicated by the manufacturers protocol. Briefly, serum-starved cells were stimulated with EphB2-Fc and then lysed using MLB lysis buffer in the presence of protease inhibitors for 15 min. Rhotekin-agarose or PAK1-agarose was added to cell lysates at a 1:1 volume ratio and incubated at 4 °C for 1 h. After brief centrifugation, the bead pellets were washed with MLB buffer. Laemmli sample buffer was added to the samples and separated by 15% SDS–PAGE, and immunoblotted with anti-RhoA, anti-Rac1 or anti-Cdc42 antibody.
Cho H.J., Hwang Y.S., Yoon J., Lee M., Lee H.G, & Daar I.O. (2017). EphrinB1 promotes cancer cell migration and invasion through the interaction with RhoGDI1. Oncogene, 37(7), 861-872.
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6

Brain Ischemia Dephosphorylation and RhoA Activity

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Dephosphorylation studies were performed with protein homogenates prepared without phosphatase inhibitors according to our previous report (Sabirzhanova et al., 2013 (link)). Equal protein amounts of sham cortical tissues samples (25 µg/25µl) were incubated at 37°C for 1 h in the presence or absence of calf intestinal alkaline phosphatase (CIP) (New England Biolabs, MA), and then were subjected to Western blot analysis with GEF-H1 antibody or phospho-GEF-H1 (Ser885).
A pull-down assay was carried out to determine RhoA GTPase activity in the brain tissue samples after brain ischemia according to the method described previously (Sabirzhanova et al., 2013 (link)). Briefly, the cortical tissues of sham-operated rats and rats subjected to ischemia followed by 0.5 h of reperfusion were homogenized in a lysis buffer, and then cleared by 10 min centrifugation at 13,000 g at 4°C (Sabirzhanova et al., 2013 (link)). The supernatants were incubated with Rho assay reagent (Cat# 14-382) (Millipore, Billerica, MA, USA). The RhoA binding beads were collected by centrifugation and then washed three times with lysis buffer. The bead-binding complexes as well as homogenates aliquots were then subjected to Western blot analysis to determine the amount of the GTP-form of RhoA, as well as the total RhoA with antibody.
Luo T., Roman P., Liu C., Sun X., Park Y, & Hu B. (2014). Upregulation of the GEF-H1 Pathway after Transient Cerebral Ischemia. Experimental neurology, 263, 306-313.
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7

RhoA Activation Assay in BMSCs

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RhoA activation was assessed using the Rho Assay Reagent (Millipore) according to the manufacturer's procedure and as described previously [19 (link)]. Briefly, human BMSCs were grown to approximately 80% confluence on 100 mm dishes and serum-starved in MEM α for 12 h and then treated with or without the above-described S1PRs antagonists before they were stimulated with 1 μM S1P. Immediately after stimulation for the indicated time, cells were rinsed with cold PBS, lysed in 300 μL of lysis buffer (25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10 mM MgCl2, 1 mM EDTA, 2% glycerol, 25 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 μg/mL aprotinin/leupeptin), and then briefly centrifuged to remove cell debris. Cell lysates were pulled down by incubation at 4°C with 20 μg of recombinant Rhotekin-binding domain bound to glutathione-agarose beads for 1 h. Following washing, bound Rho was eluted by SDS sample buffer. The eluted samples and the total cell lysates were then subjected to western blot analysis with RhoA antibody to detect active and total RhoA, respectively.
Kong Y., Wang H., Lin T, & Wang S. (2014). Sphingosine-1-phosphate/S1P Receptors Signaling Modulates Cell Migration in Human Bone Marrow-Derived Mesenchymal Stem Cells. Mediators of Inflammation, 2014, 565369.
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8

Rho GTPase Activity Assay

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GTP-bound Rho was assayed with Rho assay reagent (Millipore) by lysis in Rho buffer (25 mM Hepes, pH 7.5, 150 mM NaCl, 1% Igepal CA630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol) plus protease inhibitors. Five hundred μg of cleared lysate were incubated with 25 μg of Rho assay slurry containing Rhotekin RBD agarose or GST-bound Sepharose, at 4°C for 1 h. Beads were washed 3 times in Rho buffer and suspended in 2x Laemmli buffer. GTP-bound RhoA was identified by immunoblot.
De Marco C., Malanga D., Rinaldo N., De Vita F., Scrima M., Lovisa S., Fabris L., Carriero M.V., Franco R., Rizzuto A., Baldassarre G, & Viglietto G. (2015). Mutant AKT1-E17K is oncogenic in lung epithelial cells. Oncotarget, 6(37), 39634-39650.
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9

RhoGTPases Activity Assay Protocol

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Rho assay reagent (Millipore, 14-383) and Rac/Cdc42 assay reagent (Millipore, 14-325) were used for the RhoGTPases activity assay by the manufacturer's instructions. Cells were lysed with ice-cold MLB lysis buffer. Activation levels of RhoA, Rac1, and Cdc42 were assessed by pull-down assay using Rhotekin-agarose beads or PAK1-agarose beads and analyzed by immunoblotting using respective antibodies to RhoGTPases.
Lim J., Hwang Y.S., Yoon H.R., Yoo J., Yoon S.R., Jung H., Cho H.J, & Lee H.G. (2024). PLK1 phosphorylates RhoGDI1 and promotes cancer cell migration and invasion. Cancer Cell International, 24, 73.
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10

Quantifying Rho GTPase Activities

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RhoA, Rac1 and Cdc42 activities in cells were measured using the Rac1/Cdc42 activation Assay Kit and Rho assay reagent (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. Cells were seeded in 100 mm dishes, cultured to 60–80% confluence, and serum-starved for 6 h. The cells were washed with ice-cold PBS and lysed with cell lysis buffer. Lysates were harvested and incubated with 10–20 μg Rhotekin-RBD or PAK-PBD protein agarose beads for 1 h at 4 °C. Pellets were washed four times and subjected to Western blot analysis using anti-RhoA, anti-Rac1 and Cdc42 antibodies.
Kim N., Kim S., Lee M.W., Jeon H.J., Ryu H., Kim J.M, & Lee H.J. (2021). MITF Promotes Cell Growth, Migration and Invasion in Clear Cell Renal Cell Carcinoma by Activating the RhoA/YAP Signal Pathway. Cancers, 13(12), 2920.
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