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17-441 Sigma-AldrichRac1/Cdc42 Activation Assay Kit

The Rac1/Cdc42 Activation Assay provides an effective method for detecting Rac & Cdc42 activity in cell lysates.

Rac1/Cdc42 Activation Assay Kit MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.

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Key Applications: Affinity Binding Assay, G-Protein Assay, Activity Assay

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17-441

30 assays

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Species Reactivity	Key Applications
H, M, R	ABA, GPA, ACT

Description
Catalogue Number	17-441
Brand Family	Upstate
Trade Name	Upstate
Description	Rac1/Cdc42 Activation Assay Kit
Overview	The Rac1/Cdc42 Activation Assay (Cat. No. 17-441) provides an effective method for detecting Rac and Cdc42 activity in cell lysates. This assay uses the downstream effector of Rac/Cdc42, p21-activated protein kinase (PAK1), to isolated the active GTP-bound form of Rac/Cdc42 from the sample. The p21 binding domain (PBD) of PAK1 is expressed as a GST-fusion protein and coupled to agarose beads. After precipitation, an immunoblot is performed and the activated Rac/Cdc42 is detected with specific monoclonal antibodies, followed by HRP-conjugated secondary antibody and ECL reagent.
Materials Required but Not Delivered	stimulated and non-stimulated cell lysates pipettes, micropipettes and tips electrophoresis and immunoblotting equipment microcentrifuge microcentrifuge tubes Rac activators glutathione agarose tissue culture supplies 4C tube rocker 30C incubator or water bath glycerol 500 mM EDTA

References

Product Information
Components	Rac/cdc42 Assay Reagent (PAK-1 PBD, agarose), Catalog # 14-325 :One vial containing 300 μg of PAK-1 PBD bound to 150 μL of glutathione agarose beads, provided as a 50% slurry in 20mM PBS, pH 7.4, containing 50% glycerol for a final volume of 300 μL. Anti-Rac1, clone 23A8, Catalog #05-389: One vial containing 250 μg of protein G purified mouse IgG_2b in 250 μL of storage buffer (0.1 M Tris-glycine, pH 7.4, 0.15 M NaCl, containing 0.05% sodium azide). Anti-cdc42, (mouse monoclonal IgG₁) , Catalog # 05-542: One vial containing 50 μg of purified mouse IgG₁ in 200 μL of 50% storage buffer (20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1.5 mM sodium azide containing, 1 mg/mL BSA) and 50% glycerol. Mg2+ Lysis/Wash Buffer, 5X, Catalog # 20-168 : Two vials, each vial containing 18 mL of 5X MLB: 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl₂, 5 mM EDTA and 10% glycerol. 100X GTPγS, 10mM, Catalog # 20-176: One vial containing 50 μL of 10 mM GTPγS, 100X stock, in 50 mM Tris-HCl, pH 7.8, non-hydrolyzable analog of GTP. Sufficient to label 5 mL of cell lysates. 100X GDP, 100mM, Catalog # 20-177: One vial containing 50 μL of 100 mM GDP, 100X stock, in 50 mM Tris-HCl, pH 7.8. GDP (Guanosine 5'-Diphosphate) for in vitro labeling of G-proteins in the inactive form. Sufficient to label 5ml of cell lysates.
Quality Level	MQ100

Product Information

Components

Rac/cdc42 Assay Reagent (PAK-1 PBD, agarose), Catalog # 14-325 :One vial containing 300 μg of PAK-1 PBD bound to 150 μL of glutathione agarose beads, provided as a 50% slurry in 20mM PBS, pH 7.4, containing 50% glycerol for a final volume of 300 μL.
Anti-Rac1, clone 23A8, Catalog #05-389: One vial containing 250 μg of protein G purified mouse IgG_2b in 250 μL of storage buffer (0.1 M Tris-glycine, pH 7.4, 0.15 M NaCl, containing 0.05% sodium azide).
Anti-cdc42, (mouse monoclonal IgG₁) , Catalog # 05-542: One vial containing 50 μg of purified mouse IgG₁ in 200 μL of 50% storage buffer (20 mM sodium phosphate, pH 7.5, 150 mM NaCl, 1.5 mM sodium azide containing, 1 mg/mL BSA) and 50% glycerol.
Mg2+ Lysis/Wash Buffer, 5X, Catalog # 20-168 : Two vials, each vial containing 18 mL of 5X MLB: 125 mM HEPES, pH 7.5, 750 mM NaCl, 5% Igepal CA-630, 50 mM MgCl₂, 5 mM EDTA and 10% glycerol.
100X GTPγS, 10mM, Catalog # 20-176: One vial containing 50 μL of 10 mM GTPγS, 100X stock, in 50 mM Tris-HCl, pH 7.8, non-hydrolyzable analog of GTP. Sufficient to label 5 mL of cell lysates.
100X GDP, 100mM, Catalog # 20-177: One vial containing 50 μL of 100 mM GDP, 100X stock, in 50 mM Tris-HCl, pH 7.8. GDP (Guanosine 5'-Diphosphate) for in vitro labeling of G-proteins in the inactive form. Sufficient to label 5ml of cell lysates.

Quality Level

MQ100

Applications
Application	The Rac1/Cdc42 Activation Assay provides an effective method for detecting Rac & Cdc42 activity in cell lysates.
Key Applications	Affinity Binding Assay G-Protein Assay Activity Assay

Biological Information
Species Reactivity	Human Mouse Rat
Species Reactivity Note	Rac/cdc42 Assay Reagent (PAK-1 PBD, agarose): Species Cross-reactivity: Human and mouse. Predicted to cross-react with all mammalian species. Anti-Rac1, clone 23A8: Species Cross-reactivity: Human, mouse and rat. Other species cross-reactivity unknown. Anti-cdc42 Species Cross-reactivity: Human, mouse, rat and dog. Other species cross-reactivity unknown.
Entrez Gene Number	NM_006908.3 NM_018890.2 NM_198829.1
Entrez Gene Summary	The protein encoded by this gene is a GTPase which belongs to the RAS superfamily of small GTP-binding proteins. Members of this superfamily appear to regulate a diverse array of cellular events, including the control of cell growth, cytoskeletal reorganization, and the activation of protein kinases. Several alternatively spliced transcript variants of this gene have been described, but the full-length nature of some of these variants has not been determined.
Gene Symbol	RAC1 MGC111543 ORFNames=MIG5 PAK1 MIG5 TC-25 p21-Rac1
UniProt Number	P63000
UniProt Summary	FUNCTION: SwissProt: P63000 # Isoform B has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored partially by GTPase-activating proteins. It is able to bind to the GTPase-binding domain of PAK but not full-length PAK in a GTP- dependent manner, suggesting that the insertion does not completely abolish effector interaction. SIZE: 192 amino acids; 21450 Da SUBUNIT: Interacts with the GEF proteins PREX1, RASGRF2, DOCK1, DOCK2 and DOCK7, which promote the exchange between GDP and GTP, and therefore activate it. Interacts with PARD6A, PARD6B and PARD6G in a GTP-dependent manner. Part of a quaternary complex containing PARD3, some PARD6 protein (PARD6A, PARD6B or PARD6G) and some atypical PKC protein (PRKCI or PRKCZ), which plays a central role in epithelial cell polarization. Found in a trimeric complex composed of DOCK1 and ELMO1, which plays a central role in phagocytosis of apoptotic cells. Interacts with RALBP1 via its effector domain. Interacts with PLXNB1. Part of a complex with MAP2K3, MAP3K3, CCM2 and DEF6. Interacts with BAIAP2, CYFIP1/SRA-1 and DEF6. Interacts with Y.pseudotuberculosis YPKA and PLCB2. SUBCELLULAR LOCATION: Cell membrane; Lipid-anchor; Cytoplasmic side (By similarity). Melanosome. Note=Inner surface of plasma membrane possibly with attachment requiring prenylation of the C- terminal cysteine (By similarity). Identified by mass spectrometry in melanosome fractions from stage I to stage IV. TISSUE SPECIFICITY: Isoform B is predominantly identified in skin and epithelial tissues from the intestinal tract. The expression of isoform B is elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. DOMAIN: SwissProt: P63000 The effector region mediates interaction with DEF6. SIMILARITY: Belongs to the small GTPase superfamily. Rho family.

Physicochemical Information

Dimensions

Materials Information

Toxicological Information

Safety Information according to GHS

Safety Information

Product Usage Statements
Quality Assurance	Routinely evaluated by precipitating Rac1-GTP and cdc42-GTP from 3T3/A31 and HeLa cell lysates that had been loaded with GTPγS(lysates were loaded in vitro with GTPγS for cdc42-GTP). The precipitated Rac1-GTP and cdc42-GTP was detected by immunoblot analysis using anti-Rac1 (1 μg/ml).
Usage Statement	Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

Storage and Shipping Information
Storage Conditions	1 year at -20°C from date of shipment

Packaging Information
Material Size	30 assays

Transport Information

Supplemental Information

Specifications

Global Trade Item Number
Catalog Number	GTIN
17-441	04053252338700

Documentation

Rac1/Cdc42 Activation Assay Kit SDS

Title
Safety Data Sheet (SDS)

Rac1/Cdc42 Activation Assay Kit Certificates of Analysis

Title	Lot Number
Rac1/Cdc42 Activation Assay Kit (non-radioactive) - 2382141	2382141
Rac1/Cdc42 Activation Assay Kit (non-radioactive)	2964202
Rac1/Cdc42 Activation Assay Kit (non-radioactive)	3028337
Rac1/Cdc42 Activation Assay Kit (non-radioactive)	3036789
Rac1/Cdc42 Activation Assay Kit (non-radioactive) - 2210399	2210399
Rac1/Cdc42 Activation Assay Kit (non-radioactive) - 2294337	2294337
Rac1/Cdc42 Activation Assay Kit (non-radioactive) - 3324457	3324457
Rac1/Cdc42 Activation Assay Kit (non-radioactive) -2483957	2483957
Rac1/Cdc42 Activation Assay Kit - 2430513	2430513
Rac1/Cdc42 Activation Assay Kit - 1971849	1971849

User Guides

Title
Rac1/Cdc42 Activation Assay Kit (non-radioactive)

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