Product Uses Include
- Analysis of in vivo Cdc42 activation.
IntroductionThe 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.
Kit contentsThe 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. # BK034. The following components are included:
- GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
- Cdc42 monoclonal antibody (Cat. # ACD03)
- His-tagged Cdc42 protein (Cat. # CD01)
- GTPγS: (non-hydrolyzable GTP analog) (Cat. # BS01)
- GDP
- Cell lysis Buffer
- Wash Buffer
- Loading Buffer
- STOP Buffer
- Protease inhibitor cocktail (Cat. # PIC02)
- Manual with detailed protocols and extensive troubleshooting guide
Figure 1. The brightly colored glutathione agarose beads in BK034-S makes the kit easy to use.
Equipment needed
- SDS-PAGE minigel system and western blotting transfer apparatus
Example resultsThe 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-S Cdc42 activation assay. Activated Cdc42 was precipitated and detected in a Western blot using kit BK034-S. 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.
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
Sakabe et al., 2012. Age-related guanine nucleotide exchange factor, mouse Zizimin2, induces filopodia in bone marrow-derived dendritic cells. Immun. Ageing. v 9, p 2.
Nithipatikom et al., 2012. Cannabinoid Receptor Type 1 (CB1) Activation Inhibits Small GTPase RhoA Activity and Regulates Motility of Prostate Carcinoma Cells. Endocrinology. v 153, pp 29-41.
Asai et al., 2011. Involvement of Rac GTPase activation in phosphatidylcholine hydroperoxide-induced THP-1 cell adhesion to ICAM-1. Biochem. Biophys. Res. Commun. v 406, pp 273-277.
Abu-Elneel et al., 2008. A delta-Catenin Signaling Pathway Leading to dendritic protrusions. J. Biol. Chem. v 283, pp 32781-32791.Nur-E-Kamal et al., 2005. Three dimensional nanofibrillar surfaces induce activation of Rac. Biochem. Biophys. Res. Commun. v 331, pp 428-434.Slice et al., 2005. Angiotensin II and epidermal growth factor induce cyclooxygenase-2 expression in intestinal epithelial cells through small GTPases using distinct signaling pathways. J. Biol. Chem. v 280, pp 1582-1593.Tang et al., 2005. The Adapter Protein CrkII Regulates Neuronal Wiskott-Aldrich Syndrome Protein, Actin Polymerization, and Tension Development during Contractile Stimulation of Smooth Muscle. J. Biol. Chem. v 280, pp 23380-23389.Liu et al., 2004. Nucleotide exchange factor ECT2 interacts with the polarity protein complex Par6/Par3/protein kinase Cζ (PKCζ) and regulates PKCζ activity. Mol. Cell. Biol. v 24, pp 6665-6675.Sasai et al., 2004. The neurotrophin-receptor-related protein NRH1 is essential for convergent extension movements. Nat. Cell Biol. v 6, pp 741-748.Tang and Gunst, 2004. The small GTPase Cdc42 regulates actin polymerization and tension development during contractile stimulation of smooth muscle. J. Biol. Chem. v 279, pp 51722-51728.
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:
- When blotting use 70v for 45min only as the small G-proteins are very mobile.
- 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.
- Dry the PVDF membrane for 30 min after transfer and before blocking (not necessary for nitrocellulose)
- Making sure that the TBST contains 10 mM Tris, 0.05% Tween 20 and 150 mM NaCl.
- 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 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.
If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
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