Rac Activation Assay was performed as described previously (Yao and Tsirka, 2010 (link)). Specifically, microglia were treated with 10nM MCP-1 proteins. At various time points, the cells were lysed in RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% NP40, 0.25% sodium deoxycholate, 1mM PMSF, 1× protease inhibitor cocktail, 1× Na3VO4). The activated Rac1 was pulled down using the GST-PBD beads (a generous gift from Dr. J. Prives, Stony Brook University, Stony Brook, USA) and detected by immunoblotting using anti-Rac1 antibody (1:1000, Millipore). Total Rac1 was determined by immunoblotting using total cell lysates. Band intensities were quantified using Scion Image (Scion, Frederick, MD) or Odyssey Infrared Imaging system. The ratio of activated Rac1 intensity to total Rac1 intensity (A/T ratio) was used to indicate Rac1 activation. For FL- and K104Stop-MCP1, Rac1 activation (A/T ratio) was normalized to untreated controls at 0 time point. For FL-FL- and K104Stop-K104Stop-MCP1 Rac1 activation was normalized to their respective positive controls (FL Ctr and K104Stop Ctr), since the dimeric MCP1s failed to activate Rac1. Three independent experiments were performed for quantification.
Anti rac1 antibody
Manufactured by Merck Group
Sourced in United States
The Anti-Rac1 antibody is a laboratory reagent used to detect and quantify the Rac1 protein in biological samples. Rac1 is a small GTPase that plays a key role in various cellular processes, including actin cytoskeleton organization, cell migration, and signaling pathways. The antibody can be used in techniques such as Western blotting, immunoprecipitation, and immunocytochemistry to study the expression and localization of Rac1 in cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Rac1 activation assay kit, by Merck Group (4 mentions)
Phospho akt, by Cell Signaling Technology (2 mentions)
Scion image, by Techcomp Instruments (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Alexa fluor 647 antibody labeling kit, by Thermo Fisher Scientific (1 mentions)
Phospho p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Phospho pak1 2, by Cell Signaling Technology (1 mentions)
Calcein acetoxymethyl ester calcein am, by Thermo Fisher Scientific (1 mentions)
Pak1 2 3, by Cell Signaling Technology (1 mentions)
β tubulin, by Cell Signaling Technology (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
Casin, by ChemBridge (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Phospho egfr, by Cell Signaling Technology (1 mentions)
Phospho rac1 cdc42 ser71, by Cell Signaling Technology (1 mentions)
Anti egfp antibody, by Takara Bio (1 mentions)
Erk1 2, by Santa Cruz Biotechnology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
8 protocols using anti rac1 antibody
1
Rac1 Activation Assay for MCP-1
2
Measuring Rac1 Activity Using Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rac1 activity was measured using a Rac1 activation assay kit (Millipore, Billerica, MA, USA). Briefly, the protein sample concentrations were measured by using the BCA Protein Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China). Protein p21-activated protein kinase 1 (Pak 1) is able to bind active Rac1 form. The same total protein in each protein (1 mg) was mixed with 10 μl PAK-1 PBD (a GST fusion protein corresponding to the p21-binding domain (PBD) of human PAK-1) agarose beads for 1 h at 4 °C. Active (GTP-bound) Rac is specially combined with the p21-binding domain of p21-activated protein kinase 1. Precipitated GST bound Rac1 agarose beads were resuspended in 40 μL of 2 × reducing sample buffer and boiled for 5 min. The bound proteins were separated by 12% SDS-PAGE and immunoblotted using anti-Rac1 antibody (1:1000, Millipore, Billerica, MA, USA).
3
Molecular Mechanisms of Platelet Activation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chemicals and reagents were purchased either from Sigma-Aldrich (St. Louis, MO) or from specifically noted sources. CASIN and Pirl7 were obtained from Chembridge Corporation (San Diego, CA), and purified to greater than 99% by high performance liquid chromatography. Collagen was obtained from Chrono-Log Corporation (Havertown, PA).
Anti-Cdc42 antibody (#2466), PAK1/2/3 (#2604), Phospho-PAK1/2 (#2601, PAK1 Thr423/PAK2 Thr402, p44/42 MAPK (#9102 ERK1/2), Phospho-p44/42 MAPK ERK1/2 (#4370, Thr202/Tyr204), Akt (#9272), Phospho-Akt (#4060, Ser473), p38 MAPK (#8690, Phospho-p38 MAPK (#4511, Thr180/Tyr182), GAPDH (#2118), β-tubulin (#2128) were purchased from Cell Signaling Technology, Danvers, MA. Anti-Rac1 antibody (#05-389) was purchased from Millipore, USA. DyLight 488 anti-GPIb antibody was purchased from Emfret (Eibelstadt, Germany), calcein acetoxymethyl ester (Calcein-AM) was purchased from ThermoFisher (Grand Island, NY). Anti-mouse fibrin antibody was a kind gift from Dr. R. Camire at Children's Hospital of Philadelphia. Anti-fibrin antibody was fluorescently labeled as per manufacturer's instruction using Alexa Fluor 647- antibody labeling kit from ThermoFisher (Grand Island, NY).
Anti-Cdc42 antibody (#2466), PAK1/2/3 (#2604), Phospho-PAK1/2 (#2601, PAK1 Thr423/PAK2 Thr402, p44/42 MAPK (#9102 ERK1/2), Phospho-p44/42 MAPK ERK1/2 (#4370, Thr202/Tyr204), Akt (#9272), Phospho-Akt (#4060, Ser473), p38 MAPK (#8690, Phospho-p38 MAPK (#4511, Thr180/Tyr182), GAPDH (#2118), β-tubulin (#2128) were purchased from Cell Signaling Technology, Danvers, MA. Anti-Rac1 antibody (#05-389) was purchased from Millipore, USA. DyLight 488 anti-GPIb antibody was purchased from Emfret (Eibelstadt, Germany), calcein acetoxymethyl ester (Calcein-AM) was purchased from ThermoFisher (Grand Island, NY). Anti-mouse fibrin antibody was a kind gift from Dr. R. Camire at Children's Hospital of Philadelphia. Anti-fibrin antibody was fluorescently labeled as per manufacturer's instruction using Alexa Fluor 647- antibody labeling kit from ThermoFisher (Grand Island, NY).
4
Western Blotting and Rac1 Activation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were prepared as described previously.(26 (link)) Primary antibodies used were Phospho-EGFR (Tyr1068), Phospho-Akt (Ser473), Phospho-ERK1/2 (Thr202/Tyr204), Phospho-p38 (Thr180/Tyr182), Phospho-Rac1/cdc42 (Ser71), cyclin D1 from Cell Signaling Technology (Beverly, MA, USA), EGFR, Akt, ERK1/2, p38α, α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), an anti-RAC1 antibody from Merck Millipore, an anti-EGFP antibody from Clontech and an anti-HA antibody from Roche (Indianapolis, IN, USA). The active RAC1 was assessed using the Rac1 Activation Assay kit (Merck Millipore) according to the manufacturer's instructions. The pulled-down cell lysates were subjected to western blotting.
5
Rac1 Activation Assay in Phagocytosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were incubated with apoptotic thymocytes for 20 min at 37 °C, followed by removal of unbound targets by washing with ice-cold PBS. The cells were lysed using lysis buffer and the Rac1 pulldown assay was performed according to the manufacturer’s protocol for the Rac1 Activation Assay Kit. In this assay, the GTP-bound active form of Rac1 specifically bound to the GST-tagged PAK-1 PBD fusion protein conjugated to glutathione agarose beads. The active Rac1 was eluted from the beads and analyzed by western blotting using an anti-Rac1 antibody (05–389, Merck).
6
Rac1 Activation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rac1 activity was assessed using the Rac1 Activation Assay Kit (Millipore) according to the manufacturer’s instructions. Briefly, cell lysates were clarified by centrifugation at 14,000 ×g at 4°C for 10 min. Equal volumes of lysates were incubated with beads to pull down activated Rac1 proteins. After incubation at 4°C for 1 h, the beads were washed three times with cold MLB buffer. The Rac1 proteins were eluted with sample buffer and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Western blot was analyzed using anti-Rac1 antibodies (Millipore). GAPDH was used as a housekeeping gene for normalization.
7
RhoA and Rac1 Activation in Arthritis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine the activation of RhoA and Rac1 in FLSs of RA, OA, and patients of knee trauma, and the effect of Smo on RhoA and Rac1 activation in RA-FLSs, pull-down assays were performed according to the manufacturer’s protocol (RhoA activation assay kit and Rac1 activation assay kit, Millipore, MA, USA). Briefly, at 80% confluency, FLSs were lysed and the lysates were collected and stored at −80°C for the pull-down assay. Thirty microliters of the Rho or Rac1 Assay Reagent were added to 500 μl cell extract and the reaction mixtures were incubated for 45 min at 4°C with gentle agitation. After brief centrifugation (10 s, 14,000 × g, 4°C), the agarose beads were washed three times with 1× MLB, the supernatant was removed, and the agarose beads were re-suspended in 2× Laemmili-reducing sample buffer. Bound proteins were collected and examined by Western Blot analysis as previously described. GTP-RhoA or GTP-Rac1 was detected using anti-RhoA (3 μg/ml, Millipore, MA, USA) or anti-Rac1 antibodies (1 μg/ml, Millipore, MA, USA), respectively.
8
Endothelial Cell Growth and Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endothelial growth medium (EGM-2 BulletKit) was purchased from Clonetics (Walkersville, MD), and Vascular Cell Basal Medium (EGM Kit-VEGF) was purchased from ATCC. Phenethyl isothiocyanate (PEITC), SP600125, Hoechst 33342, protease inhibitor cocktail, and phalloidin-tetramethylrhodamine B isothiocyanate were purchased from Sigma (St. Louis, MO) and NSC-23766 from Merck Chemicals Ltd. (Darmstadt, Germany). Chemiluminescent detection reagents (ECL Plus Western Blotting) were purchased from Amersham Biosciences (Piscataway, NJ). LY294002 and antibodies to phospho-Akt (Ser473), Akt, SAPK/JNK, phospho-SAPK/JNK (Thr183/Tyr185), and phospho-FAK (Tyr397) were from Cell Signaling Technology (Danvers, MA); antibodies to BAG3 were from BIOUNIVERSA s.r.l (Montoro, AV, Italy), antibodies to PI3K, phospho-PI3K, and GAPDH from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA), and anti-Rac1 antibodies from Millipore (Bedford, MA) and Pierce (Rockford, IL). Normal goat serum, horseradish peroxidase- (HRP-) conjugated secondary antibody, enhanced chemiluminescence reagents, anti-mouse IgG, and the Dylight 488-conjugated anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (Suffolk, UK).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!