Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 50 Assays

Rac1 Activation Assay Biochem Kit (bead pull-down format) - 50 Assays
$0.00

Product Uses Include

  • Analysis of in vivo Rac1 activation levels.
  • Detection of compounds and proteins that enhance Rac1 activity
  • Detection of compounds and proteins that inhibit Rac1 activity

Introduction
The Rho switch operates by alternating between an active, GTP-bound state and an inactive, GDP-bound state.  Understanding the mechanisms that regulate activation / inactivation of the GTPases is of obvious biological significance and is a subject of intense investigation.  The fact that many Rho family effector proteins will specifically recognize the GTP bound form of the protein has been exploited experimentally to develop a powerful affinity purification assay that monitors Rac and Cdc42 protein activation.  The assay uses the Cdc42/Rac Interactive Binding (CRIB) region (also called the p21 Binding Domain, PBD) of the Cdc42 / Rac effector protein, p21 activated kinase 1 (PAK).  The CRIB/PBD protein motif has been shown to bind specifically to the GTP-bound form of Rac and/or Cdc42 proteins.  The fact that the PBD region of PAK has a high affinity for both GTP-Rac and GTP-Cdc42 and that PAK binding results in a significantly reduced intrinsic and catalytic rate of hydrolysis of both Rac and Cdc42 make it an ideal tool for affinity purification of GTP-Rac and GTP-Cdc42 from cell lysates.  The PAK-PBD protein supplied in this kit corresponds to residues 67-150.  This includes the highly conserved CRIB region (aa 74-88) plus sequences required for the high affinity interaction with GTP-Rac and GTP-Cdc42.  The PAK-PBD is in the form of a GST fusion protein, which allows one to "pull-down" the PAK-PBD/GTP-Rac (or GTP-Cdc42) complex with glutathione affinity beads.  The assay therefore provides a simple means of quantitating Rac or Cdc42 activation in cells.  The amount of activated Rac is determined by a Western blot using a Rac-specific antibody.



Kit contents
The kit contains sufficient materials for 50 assays, depending on assay setup, and includes reagents for positive and negative controls. The following components are included:

  1. GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
  2. Rac1 monoclonal antibody (Cat. # ARC03)
  3. His-tagged Rac1 protein (Cat. # RC01)
  4. GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
  5. GDP
  6. Cell lysis Buffer
  7. Wash Buffer
  8. Loading Buffer
  9. STOP Buffer
  10. Protease inhibitor cocktail (Cat. # PIC02)
  11. Manual with detailed protocols and extensive troubleshooting guide
beads

Figure 1.  The brightly colored glutathione agarose beads in BK035 makes the kit easy to use.

Equipment needed

  1. SDS-PAGE minigel system and western blotting transfer apparatus

Example results
The Rac1 activation assay was tested by loading the Rac1 protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded Rac1 is very efficiently precipitated while very little GDP-loaded Rac1 is precipitated (Fig. 2).

bk034fig2

Figure 2. Results from BK035 Rac1 activation assay. Activated Rac1 was precipitated and detected in a Western blot using kit BK035. The first lane shows a 50 ng recombinant His-tagged Rac1 standard (Recombinant His-Rac1). The following lanes shows the pull-down of inactive, GDP-loaded Rac1 (Rac1-GDP PD) or active, GTPγS-loaded Rac1 (Rac1-GTP PD) from equal amounts of cell lysates.

Please check out the new version of the Rac Activation Assay and associated products:

G-LISA Products:
Cdc42 G-LISA™ Activation Assay, colorimetric format (Cat.# BK127)
Rac1 G-LISA™ Activation Assay, luminescence format (Cat.# BK126)
Rac1,2,3 G-LISA™ Activation Assay, colorimetric format (Cat.# BK125)
RhoA G-LISA™ Activation Assay, colorimetric format (Cat.# BK124)
RhoA G-LISA™ Activation Assay, luminescence format (Cat.# BK121)

Associated Products:
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Adan, H et al.Activated Src requires Cadherin-11, Rac, and gp130 for Stat3 activation and survival of mouse Balb/c3T3 fibroblastsCancer Gene …2022Article Link
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Bai, Xiaoyuan et al.Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfectionCell Reports2021ISSN 2211-1247
Masi, Ilenia et al.Endothelin-1 drives invadopodia and interaction with mesothelial cells through ILKCell Reports2021ISSN 2211-1247
Guo, Shuyu et al.Trio cooperates with Myh9 to regulate neural crest-derived craniofacial developmentTheranostics2021ISSN 1838-7640
Floerchinger, Alessia et al.Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imagingCell Reports2021ISSN 2211-1247
Li, Chunsen et al.GEFT Inhibits Autophagy and Apoptosis in Rhabdomyosarcoma via Activation of the Rac1/Cdc42-mTOR Signaling PathwayFrontiers in Oncology2021ISSN 2234-943X
Romano, Roberta et al.Alteration of the late endocytic pathway in Charcot–Marie–Tooth type 2B diseaseCellular and Molecular Life Sciences2021ISSN 1420-9071
Wang, Junyi et al.Rho-GEF Trio regulates osteosarcoma progression and osteogenic differentiation through Rac1 and RhoACell Death and Disease2021ISSN 2041-4889
Zhou, Wenqing et al.Mitofusin 2 regulates neutrophil adhesive migration and the actin cytoskeletonJournal of Cell Science2021ISSN 1477-9137
Pickering, K. A. et al.A RAC-GEF network critical for early intestinal tumourigenesisNature Communications2021ISSN 2041-1723
Agbaegbu Iweka, Chinyere et al.The lipid phosphatase-like protein PLPPR1 associates with RhoGDI1 to modulate RhoA activation in response to axon growth inhibitory moleculesJournal of Neurochemistry2021ISSN 1471-4159
McCray, Brett A. et al.Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extensionNature Communications2021ISSN 2041-1723
Wang, Jiang Lin et al.Spinophilin modulates pain through suppressing dendritic spine morphogenesis via negative control of Rac1-ERK signaling in rat spinal dorsal hornNeurobiology of Disease2021ISSN 1095-953X
Li, Zean et al.The metastatic promoter DEPDC1B induces epithelial‐mesenchymal transition and promotes prostate cancer cell proliferation via Rac1‐PAK1 signalingClinical and Translational Medicine2020ISSN 2001--1326
Ichikawa, Takehiko et al.Non-junctional role of Cadherin3 in cell migration and contact inhibition of locomotion via domain-dependent, opposing regulation of Rac1Scientific Reports2020ISSN 2045-2322
Danelon, Victor et al.Modular and Distinct Plexin-A4/FARP2/Rac1 Signaling Controls Dendrite MorphogenesisJournal of Neuroscience2020ISSN 1529-2401
Moody, Jasmine C. et al.The Rho-GEF PIX-1 directs assembly or stability of lateral attachment structures between muscle cellsNature Communications2020ISSN 2041-1723
Wei, Yiju et al. NEDD 4L‐mediated Merlin ubiquitination facilitates Hippo pathway activation EMBO reports2020ISSN 1469--221X
Aladowicz, Ewa et al.Shcd binds dock4, promotes ameboid motility and metastasis dissemination, predicting poor prognosis in melanomaCancers2020ISSN 2072-6694
Barabutis, Nektarios et al.Protective mechanism of the selective vasopressin V1A receptor agonist selepressin against endothelial barrier dysfunctionJournal of Pharmacology and Experimental Therapeutics2020ISSN 1521-0103
Benz, Peter M. et al.AKAP12 deficiency impairs VEGF-induced endothelial cell migration and sproutingActa physiologica (Oxford, England)2020ISSN 1748--1716
Joshi, Rakesh et al.DLC1 SAM domain-binding peptides inhibit cancer cell growth and migration by inactivating RhoAJournal of Biological Chemistry2020ISSN 1083-351X
Larribère, Lionel et al.NF1-RAC1 axis regulates migration of the melanocytic lineageTranslational Oncology2020ISSN 1936-5233
Lian, Eric Y. et al.RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinomaOncogene2020ISSN 1476-5594
D'Amore, Claudio et al.“Janus” efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanismsBiochimica et Biophysica Acta - Molecular Cell Research2020ISSN 1879-2596
Smalley, Tracess et al.The Atypical Protein Kinase C Small Molecule Inhibitor ζ-Stat, and Its Effects on Invasion Through Decreases in PKC-ζ Protein ExpressionFrontiers in Oncology2020ISSN 2234-943X
Fulmer, Diana et al.Desert hedgehog-primary cilia cross talk shapes mitral valve tissue by organizing smooth muscle actinDevelopmental Biology2020ISSN 1095-564X
Thamilselvan, Vijayalakshmi et al.P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-CellsCellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology2020ISSN 1421--9778
Won, So Yeon et al.Fibulin 5, a human Wharton's jelly-derived mesenchymal stem cells-secreted paracrine factor, attenuates peripheral nervous system myelination defects through the Integrin-RAC1 signaling axisStem Cells2020ISSN 1549-4918
Shin, Seung Kak et al.Exogenous 8-hydroxydeoxyguanosine ameliorates liver fibrosis through the inhibition of Rac1-NADPH oxidase signalingJournal of gastroenterology and hepatology2020ISSN 1440--1746
Wang, Ruixiao et al.Rac1 silencing, NSC23766 and EHT1864 reduce growth and actin organization of bladder smooth muscle cellsLife Sciences2020ISSN 1879-0631
Chen, Lixia et al.CSRP2 suppresses colorectal cancer progression via p130Cas/Rac1 axis-meditated ERK, PAK, and HIPPO signaling pathwaysTheranostics2020ISSN 1838-7640
Dagliyan, Onur et al.Engineering proteins for allosteric control by light or ligandsNature Protocols2019ISSN 1750-2799
Zhou, Yi Fan et al.Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic strokeFASEB Journal2019ISSN 1530-6860
Lang, Yue et al.MiR-30 family prevents uPAR-ITGB3 signaling activation through calcineurin-NFATC pathway to protect podocytesCell Death and Disease2019ISSN 2041-4889
Park, Jin Seok et al.Switch-like enhancement of epithelial-mesenchymal transition by YAP through feedback regulation of WT1 and Rho-family GTPasesNature Communications2019ISSN 2041-1723
Liu, Chunxia et al.Epigenetically upregulated GEFT-derived invasion and metastasis of rhabdomyosarcoma via epithelial mesenchymal transition promoted by the Rac1/Cdc42-PAK signalling pathwayEBioMedicine2019ISSN 2352-3964
Tsygankova, Oxana M. et al.A unique role for clathrin light chain A in cell spreading and migrationJournal of Cell Science2019ISSN 1477-9137
Amato, Clelia et al.WASP Restricts Active Rac to Maintain Cells’ Front-Rear PolarizationCurrent Biology2019ISSN 0960-9822
Yu, Qingfeng et al.A NAV2729-sensitive mechanism promotes adrenergic smooth muscle contraction and growth of stromal cells in the human prostateJournal of Biological Chemistry2019ISSN 1083-351X
Wong, Bin Sheng et al.A direct podocalyxin–dynamin-2 interaction regulates cytoskeletal dynamics to promote migration and metastasis in pancreatic cancer cellsCancer Research2019ISSN 1538-7445
Shi, Hao et al.Hippo Kinases Mst1 and Mst2 Sense and Amplify IL-2R-STAT5 Signaling in Regulatory T Cells to Establish Stable Regulatory ActivityImmunity2018ISSN 1097-4180
Hayashi, Kentaro et al.Intracellular calcium signal at the leading edge regulates mesodermal sheet migration during Xenopus gastrulationScientific Reports2018ISSN 2045-2322
Mills, Shirley C. et al.Rac1 plays a role in CXCL12 but not CCL3-induced chemotaxis and Rac1 GEF inhibitor NSC23766 has off target effects on CXCR4Cellular Signalling2018ISSN 1873-3913
Murillo, Miguel M. et al.Disruption of the Interaction of RAS with PI 3-Kinase Induces Regression of EGFR-Mutant-Driven Lung CancerCell Reports2018ISSN 2211-1247
Shang, Wanjing et al.Genome-wide CRISPR screen identifies FAM49B as a key regulator of actin dynamics and T cell activationProceedings of the National Academy of Sciences of the United States of America2018ISSN 1091-6490
Liu, Yunlong et al.Social Isolation Induces Rac1-Dependent Forgetting of Social MemoryCell Reports2018ISSN 2211-1247
Yang, Huan et al.Cytotoxic Necrotizing Factor 1 Downregulates CD36 Transcription in Macrophages to Induce Inflammation During Acute Urinary Tract InfectionsFrontiers in Immunology2018ISSN 1664-3224
McQueeney, Kelley E. et al.Targeting ovarian cancer and endothelium with an allosteric PTP4A3 phosphatase inhibitorOncotarget2018ISSN 1949-2553
Zago, Giulia et al.Ralb directly triggers invasion downstream ras by mobilizing the wave complexeLife2018ISSN 2050-084X
Wu, Yuqing et al.Mycobacterial infection is promoted by neutral sphingomyelinase 2 regulating a signaling cascade leading to activation of β1-integrinCellular Physiology and Biochemistry2018ISSN 1421-9778
Barabutis, Nektarios et al.Wild-type p53 enhances endothelial barrier function by mediating RAC1 signalling and RhoA inhibitionJournal of Cellular and Molecular Medicine2018ISSN 1582-1838
Hayes, Madeline N. et al.Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in RhabdomyosarcomaCell Stem Cell2018ISSN 1875-9777
Herlemann, Annika et al.Inhibition of smooth muscle contraction and ARF6 activity by the inhibitor for cytohesin GEFs, secinH3, in the human prostateAmerican Journal of Physiology - Renal Physiology2018ISSN 1522-1466
Yao, Zhihui et al.P311 Accelerates Skin Wound Reepithelialization by Promoting Epidermal Stem Cell Migration Through RhoA and Rac1 ActivationStem Cells and Development2017ISSN 1557-8534
Tarr, Joseph T. et al.The pivotal role of CCN2 in mammalian palatogenesisJournal of Cell Communication and Signaling2017ISSN 1873-961X
Gao, Jie et al.The E3 ubiquitin ligase IDOL regulates synaptic ApoER2 levels and is important for plasticity and learningeLife2017ISSN 2050-084X
Margiotta, Azzurra et al.Rab7a regulates cell migration through Rac1 and vimentinBiochimica et Biophysica Acta - Molecular Cell Research2017ISSN 1879-2596
Guo, Yaxiu et al.Cytotoxic necrotizing factor 1 promotes prostate cancer progression through activating the Cdc42–PAK1 axisJournal of Pathology2017ISSN 1096-9896
Tormos, Ana M. et al.P38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and agingPLoS ONE2017ISSN 1932-6203
Barrera-Chimal, Jonatan et al.Benefit of mineralocorticoid receptor antagonism in AKI: Role of vascular smooth muscle Rac1Journal of the American Society of Nephrology2017ISSN 1533-3450
Tie, S. R. et al.Regulation of sarcoma cell migration, invasion and invadopodia formation by AFAP1L1 through a phosphotyrosine-dependent pathwayOncogene2016ISSN 1476-5594
Han, Jie et al.Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive ratsMolecular Medicine Reports2016ISSN 1791-3004
Wang, Yan et al.Involvement of Rac1 signalling pathway in the development and maintenance of acute inflammatory pain induced by bee venom injectionBritish Journal of Pharmacology2016ISSN 1476-5381
Zhan, Rixing et al.Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signallingScientific Reports2016ISSN 2045-2322
Wang, Shi Jie et al.CD147 promotes Src-dependent activation of Rac1 signaling through STAT3/DOCK8 during the motility of hepatocellular carcinoma cellsOncotarget2015ISSN 1949-2553
Zhan, Rixing et al.Nitric oxide enhances keratinocyte cell migration by regulating Rho GTPase via cGMP-PKG signallingPLoS ONE2015ISSN 1932-6203
Wang, Y. et al.Inhibition of prostate smooth muscle contraction and prostate stromal cell growth by the inhibitors of Rac, NSC23766 and EHT1864British Journal of Pharmacology2015ISSN 1476-5381
Tang, Xiaoyun et al.Lipid phosphate phosphatase-1 expression in cancer cells attenuates tumor growth and metastasis in miceJournal of Lipid Research2014ISSN 1539-7262
Hara, Yusuke et al.Directional migration of leading-edge mesoderm generates physical forces: Implication in Xenopus notochord formation during gastrulationDevelopmental Biology2013ISSN 1095-564X
Han, Gangwen et al.Preventive and therapeutic effects of Smad7 on radiation-induced oral mucositisNature Medicine2013ISSN 1078-8956
Zallocchi, Marisa et al.α1β1 Integrin/rac1-dependent mesangial invasion of glomerular capillaries in alport syndromeAmerican Journal of Pathology2013ISSN 0002-9440
Geletu, M. et al.Classical cadherins control survival through the gp130/Stat3 axisBiochimica et Biophysica Acta - Molecular Cell Research2013ISSN 0167-4889
Gastonguay, Adam et al.The role of Rac1 in the regulation of NF-kB activity, cell proliferation, and cell migration in non-small cell lung carcinomaCancer Biology & Therapy2012ISSN 1538--4047
Wong, Hon Kit et al.Merlin/NF2 regulates angiogenesis in schwannomas through a Rac1/semaphorin 3F-dependent mechanismNeoplasia2012ISSN 1476-5586
Ninkovi, Jana et al.Morphine decreases bacterial phagocytosis by inhibiting actin polymerization through cAMP-, Rac-1-, and p38 MAPK-dependent mechanismsAmerican Journal of Pathology2012ISSN 0002-9440
Nithipatikom, Kasem et al.Cannabinoid receptor type 1 (CB1) activation inhibits small GTPase RhoA activity and regulates motility of prostate carcinoma cellsEndocrinology2012ISSN 1945--7170
Syed, Ismail et al.L-threo-C 6 -pyridinium-ceramide bromide, a novel cationic ceramide, induces NADPH oxidase activation, mitochondrial dysfunction and loss in cell viability in INS 832/13 β-cellsCellular Physiology and Biochemistry2012ISSN 1421-9778
Lee, Wonhwa et al.Barrier protective effects of withaferin A in HMGB1-induced inflammatory responses in both cellular and animal modelsToxicology and applied pharmacology2012ISSN 1096--0333
Ock, Chan Young et al.A novel approach for stress-induced gastritis based on paradoxical anti-oxidative and anti-inflammatory action of exogenous 8-hydroxydeoxyguanosineBiochemical pharmacology2011ISSN 1873--2968
Garkavtsev, Igor et al.Dehydro-α-lapachone, a plant product with antivascular activityProceedings of the National Academy of Sciences of the United States of America2011ISSN 0027-8424
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Jayaram, Bhavaani et al.Isoprenylcysteine carboxyl methyltransferase facilitates glucose-induced Rac1 activation, ROS generation and insulin secretion in INS 832/13 β-cellsIslets2011ISSN 1938-2014
Souma, Tomokazu et al.Luminal alkalinization attenuates proteinuria-induced oxidative damage in proximal tubular cellsJournal of the American Society of Nephrology2011ISSN 1046-6673
Kobayashi, Takashi et al.Activation of Rac1 is closely related to androgen-independent cell proliferation of prostate cancer cells both in vitro and in vivoMolecular Endocrinology2010ISSN 0888-8809
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Question 1: I have high background and/or multiple bands on my western blot.  How can I fix this?

Answer 1:  There are multiple causes of high background and/or multiple bands.  Some suggestions to improve background signal include:

  1. When blotting use 70v for 45min only as the small G-proteins are very mobile.
  2. Fully remove SDS from the gel by using a non-SDS containing buffer for transfer and performing a full 15 min gel wash step in the transfer buffer before blotting.
  3. Dry the PVDF membrane for 30 min after transfer and before blocking (not necessary for nitrocellulose)
  4. Making sure that the TBST contains 10 mM Tris, 0.05% Tween 20 and 150 mM NaCl.
  5. Incubating with the primary antibody overnight at 4°C and using the appropriate ECL detection system.  

 

Question 2: How much of the beads should I use for my pull-down experiments?

Answer 2:  PAK-PBD-GST beads (Cat. # PAK02) will bind to Rac1-GDP with a much lower affinity than Rac1-GTP.  If too many PAK-PBD beads are added to the pull-down assay, there will be significant binding to inactive (GDP-bound) Rac1.  The result of this will be an underestimation of Rac1 activation.  For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Rac1 activation or inactivation assay.  Once optimal conditions have been established, bead titrations should no longer be necessary.  We recommend 10, 15 and 20 μg bead titrations.

 

Question 3:  How can I test whether the beads are working properly?

Answer 3:  A standard biological assay for PAK-PBD GST protein beads consists of a Rac protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP.  Here are guidelines to follow (see Cat. # PAK02 and BK035 datasheets for more details):

 

Positive Cellular Protein Control:

Total cell lysate (300 – 800 μg) should be loaded with GTPγS as a positive control for the pull-down assay.  The following reaction details how to load endogenous Rac1 with the nonhydrolysable GTP analog (GTPγS).  This is an excellent substrate for PAK-PBD beads and should result in a strong positive signal in a pull-down assay.

 

a) Perform GTP loading on 300 – 800 μg of cell lysate (0.5 mg/ml protein concentration) by adding 1/10th volume of Loading Buffer.

b) Immediately add 1/100th volume of GTPγS (200 μM final concentration). Under these conditions 5 - 10% of the Rac1 protein will load with non-hydrolysable GTPγS and will be “pulled-down” with the PAK-PBD beads in the assay.

c) Incubate the control sample at 30°C for 15 min with gentle rotation.

d) Stop the reaction by transferring the tube to 4°C and adding 1/10th volume of STOP Buffer.

e) Use this sample immediately in a pull-down assay.

 

Negative Cellular Protein Control:

This reaction should be performed in an identical manner to the Positive Control reaction except that 1/100th volume of GDP (1 mM final concentration) should be added to the reaction in place of the GTPγS.  Loading endogenous Rac1 with GDP will inactivate Rac1 and this complex will bind very poorly to PAK-PBD beads.

 

 

If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com