Product Uses Include
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-Cdc42 (or GTP-Rac) complex with glutathione affinity beads. The assay therefore provides a simple means of quantitating Rac/Cdc42 activation in cells. The amount of activated Cdc42 is determined by a Western blot using a Cdc42 specific antibody.
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:
Figure 1. The brightly colored glutathione agarose beads in BK034 makes the kit easy to use.
The Cdc42 activation assay was tested by loading the Cdc42 protein in cell lysates with either GTPγS or GDP. As expected, the GTPγS-loaded Cdc42 is very efficiently precipitated while very little GDP-loaded Cdc42 is precipitated (Fig. 2).
Figure 2. Results from BK034 Cdc42 activation assay. Activated Cdc42 was precipitated and detected in a Western blot using kit BK034. The first lane shows a 50 ng recombinant His-tagged Cdc42 standard (Rec. His-Cdc42). The following lanes shows the pull-down of inactive, GDP-loaded Cdc42 (Cdc42-GDP PD) or active, GTPγS-loaded Cdc42 (Cdc42-GTP PD) from equal amounts of cell lysates.
|Spel, Lotte et al.||CDC42 regulates PYRIN inflammasome assembly||Cell Reports||2022||ISSN 2211--1247|
|Khan, Alamzeb et al.||ArhGEF12 activates Rap1A and not RhoA in human dermal microvascular endothelial cells to reduce tumor necrosis factor-induced leak||FASEB journal : official publication of the Federation of American Societies for Experimental Biology||2022||ISSN 1530-6860|
|Li, Xiaopeng et al.||Hhex inhibits cell migration via regulating RHOA/CDC42-CFL1 axis in human lung cancer cells||Cell Communication and Signaling||2021||ISSN 1478-811X|
|McCray, Brett A. et al.||Neuropathy-causing TRPV4 mutations disrupt TRPV4-RhoA interactions and impair neurite extension||Nature Communications||2021||ISSN 2041-1723|
|Zhou, Yongjie et al.||Congenital biliary atresia is correlated with disrupted cell junctions and polarity caused by Cdc42 insufficiency in the liver||Theranostics||2021||ISSN 1838-7640|
|Li, Chunsen et al.||GEFT Inhibits Autophagy and Apoptosis in Rhabdomyosarcoma via Activation of the Rac1/Cdc42-mTOR Signaling Pathway||Frontiers in Oncology||2021||ISSN 2234-943X|
|Hosseini, Kamran et al.||EMT-Induced Cell-Mechanical Changes Enhance Mitotic Rounding Strength||Advanced Science||2020||ISSN 2198-3844|
|Zhang, Jiawei et al.||In vivo and in vitro activation of dormant primordial follicles by EGF treatment in mouse and human||Clinical and Translational Medicine||2020||ISSN 2001--1326|
|Lian, Eric Y. et al.||RET isoforms contribute differentially to invasive processes in pancreatic ductal adenocarcinoma||Oncogene||2020||ISSN 1476-5594|
|Chen, Lixia et al.||CSRP2 suppresses colorectal cancer progression via p130Cas/Rac1 axis-meditated ERK, PAK, and HIPPO signaling pathways||Theranostics||2020||ISSN 1838-7640|
|Gu, Jiawen et al.||Rho-GEF trio regulates osteoclast differentiation and function by Rac1/Cdc42||Experimental Cell Research||2020||ISSN 1090-2422|
|Lang, Yue et al.||MiR-30 family prevents uPAR-ITGB3 signaling activation through calcineurin-NFATC pathway to protect podocytes||Cell Death and Disease||2019||ISSN 2041-4889|
|Liu, Chunxia et al.||Epigenetically upregulated GEFT-derived invasion and metastasis of rhabdomyosarcoma via epithelial mesenchymal transition promoted by the Rac1/Cdc42-PAK signalling pathway||EBioMedicine||2019||ISSN 2352-3964|
|Zhou, Yi Fan et al.||Sema3E/PlexinD1 signaling inhibits postischemic angiogenesis by regulating endothelial DLL4 and filopodia formation in a rat model of ischemic stroke||FASEB Journal||2019||ISSN 1530-6860|
|Dagliyan, Onur et al.||Engineering proteins for allosteric control by light or ligands||Nature Protocols||2019||ISSN 1750-2799|
|Carvalho, J. R. et al.||Non-canonical Wnt signaling regulates junctional mechanocoupling during angiogenic collective cell migration||eLife||2019||ISSN 2050-084X|
|Veluthakal, Rajakrishnan et al.||Restoration of glucose-stimulated Cdc42-PAK1 activation and insulin secretion by a selective Epac activator in type 2 diabetic human islets||Diabetes||2018||ISSN 1939-327X|
|Sepúlveda-Ramírez, Silvia P. et al.||Cdc42 controls primary mesenchyme cell morphogenesis in the sea urchin embryo||Developmental Biology||2018||ISSN 1095-564X|
|Chavali, Manideep et al.||Non-canonical Wnt signaling regulates neural stem cell quiescence during homeostasis and after demyelination||Nature Communications||2018||ISSN 2041-1723|
|Yang, Huan et al.||Cytotoxic Necrotizing Factor 1 Downregulates CD36 Transcription in Macrophages to Induce Inflammation During Acute Urinary Tract Infections||Frontiers in Immunology||2018||ISSN 1664-3224|
|Saito, Masaki et al.||Tctex‐1 controls ciliary resorption by regulating branched actin polymerization and endocytosis||EMBO reports||2017||ISSN 1469--221X|
|Tormos, Ana M. et al.||P38α regulates actin cytoskeleton and cytokinesis in hepatocytes during development and aging||PLoS ONE||2017||ISSN 1932-6203|
|Guo, Yaxiu et al.||Cytotoxic necrotizing factor 1 promotes prostate cancer progression through activating the Cdc42–PAK1 axis||Journal of Pathology||2017||ISSN 1096-9896|
|Sunkavalli, Ushasree et al.||Analysis of host microRNA function uncovers a role for miR-29b-2-5p in Shigella capture by filopodia||PLoS Pathogens||2017||ISSN 1553-7374|
|Vidal-Quadras, Maite et al.||Endocytic turnover of Rab8 controls cell polarization||Journal of Cell Science||2017||ISSN 1477-9137|
|Yao, Zhihui et al.||P311 Accelerates Skin Wound Reepithelialization by Promoting Epidermal Stem Cell Migration Through RhoA and Rac1 Activation||Stem Cells and Development||2017||ISSN 1557-8534|
|Zhan, Rixing et al.||Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling||Scientific Reports||2016||ISSN 2045-2322|
|Jia, L. et al.||KLF5 promotes breast cancer proliferation, migration and invasion in part by upregulating the transcription of TNFAIP2||Oncogene||2016||ISSN 1476-5594|
|Moodley, Serisha et al.||Stimulus-dependent dissociation between XB130 and Tks5 scaffold proteins promotes airway epithelial cell migration||Oncotarget||2016||ISSN 1949-2553|
|Zhan, Rixing et al.||Nitric oxide enhances keratinocyte cell migration by regulating Rho GTPase via cGMP-PKG signalling||PLoS ONE||2015||ISSN 1932-6203|
|Tang, Xiaoyun et al.||Lipid phosphate phosphatase-1 expression in cancer cells attenuates tumor growth and metastasis in mice||Journal of Lipid Research||2014||ISSN 1539-7262|
|Ricotti, Leonardo et al.||Boron nitride nanotube-mediated stimulation modulates F/G-actin ratio and mechanical properties of human dermal fibroblasts||Journal of Nanoparticle Research||2014||ISSN 1388-0764|
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|Sakabe, Isamu et al.||Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells||Immunity and Ageing||2012||ISSN 1742-4933|
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|Chellaiah, Meenakshi A.||Regulation of actin ring formation by Rho GTPases in osteoclasts||Journal of Biological Chemistry||2005||ISSN 0021-9258|
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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:
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 Cdc42-GDP with a much lower affinity than Cdc42-GTP. If too many PAK-PBD beads are added to the pull-down assay, there will be significant binding to inactive (GDP-bound) Cdc42. The result of this will be an underestimation of Cdc42 activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given Cdc42 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 Cdc42 protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP. Here are guidelines to follow (see Cat. # PAK02 or BK034 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 Cdc42 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 Cdc42 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 Cdc42 with GDP will inactivate Cdc42 and this complex will bind very poorly to PAK-PBD beads.
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