Rac1 Pull-down Activation Assay Biochem Kit (bead pull-down format) - 20 Assays

Rac1 Activation Assay Biochem Kit (bead pull-down format) - 20 Assays

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

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 20 assays, depending on assay setup, and includes reagents for positive and negative controls.  A larger 50 assay version of this kit is available as Cat. # BK035-S. 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

    Figure 1.  The brightly colored glutathione agarose beads in BK035-S 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).


    Figure 2. Results from BK035-S Rac1 activation assay. Activated Rac1 was precipitated and detected in a Western blot using kit BK035-S. 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

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    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
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    Li, Chunsen et al.GEFT Inhibits Autophagy and Apoptosis in Rhabdomyosarcoma via Activation of the Rac1/Cdc42-mTOR Signaling PathwayFrontiers in Oncology2021ISSN 2234-943X
    McCray, Brett A. et al.Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extensionNature Communications2021ISSN 2041-1723
    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
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    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
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    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
    Liu, Yunlong et al.Social Isolation Induces Rac1-Dependent Forgetting of Social MemoryCell Reports2018ISSN 2211-1247
    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
    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
    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
    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
    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
    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
    Guo, Yaxiu et al.Cytotoxic necrotizing factor 1 promotes prostate cancer progression through activating the Cdc42–PAK1 axisJournal of Pathology2017ISSN 1096-9896
    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
    Han, Jie et al.Farnesyl pyrophosphate synthase inhibitor, ibandronate, improves endothelial function in spontaneously hypertensive ratsMolecular Medicine Reports2016ISSN 1791-3004
    Zhan, Rixing et al.Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signallingScientific Reports2016ISSN 2045-2322
    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
    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
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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