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Cdc42 Pull-Down Activation Assay Kit

Cdc42 Pull-Down Activation Assay Kit
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$737.00

Cat.#:  80701

   Size:   30 Assays

In Stock

          Product Description          

Cdc42 Pull-Down Activation Assay Kit

Cat. # 80701

Introduction

A. Background
Small GTPases are a super-family of cellular signaling regulators. Cdc42 belongs to the Rho sub-family of GTPases that regulate cell motility, cell division, and gene transcription. GTP binding increases the activity of Cdc42, and the hydrolysis of GTP to GDP renders it inactive.
Currently the activation of Cdc42 proteins is assayed with the binding of GTP-bound Cdc42 to the p21-binding domain (PBD) of p21-activated protein kinase (PAK). This method is based on the observation that the active, GTP-bound Cdc42 (and Rac) could bind to the PBD of PAK. However, the reproducibility of this method is poor. This is partially due to the relatively quick hydrolysis of GTP to GDP during the assay procedure, and the low binding affinity of PBD to Cdc42-GTP.
The Cdc42 Activation Assay Kit is based on the configuration-specific monoclonal antibody that specifically recognizes Cdc42-GTP, but not Cdc42-GDP. Given the high affinity of monoclonal antibodies to their antigens, the activation assay could be performed in a much shorter time. This assay provides the reliable results with consistent reproducibility.
B. Assay Principle
The Cdc42 Activation Assay Kit uses configuration-specific anti-Cdc42-GTP Mouse monoclonal antibody to measure Cdc42-GTP levels in cell extracts or in vitro GTPγS loading Cdc42 activation assays. Anti-Cdc42-GTP mouse monoclonal antibody is first incubated with cell lysates containing Cdc42-GTP. Next, the GTP-bound Cdc42 is pulled down by protein A/G agarose. Finally, the precipitated Cdc42-GTP is detected through immunoblot analysis using Anti-Cdc42 Rabbit Polyclonal Antibody.
The anti-Cdc42-GTP monoclonal antibody can also be used to monitor the activation of Cdc42 in cells and in tissues by immunohistochemistry.
C. Kit Components
1. Anti-Cdc42-GTP Mouse Monoclonal Antibody (Cat. # 26905): 30 µL (1 mg/ml) in PBS, pH 7.4, containing 50% glycerol. This antibody specifically recognizes Cdc42-GTP from all vertebrates.
2. Protein A/G Agarose (Cat. # 30301): 600 µL of 50% slurry.
3. 5X Assay/Lysis Buffer (Cat. # 30302): 30 mL of 250 mM Tris-HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100.
4. Anti-Cdc42 Rabbit Polyclonal Antibody (Cat. # 21010): 50 µL (1 mg/mL) in PBS, pH 7.4, contained 50% glycerol.
5. 100X GTPγS (Cat. # 30303): 50 µl at 10 mM, use 5 µL of GTPγS for  GTP-labeling of 0.5 mL of cell lysate.
6. 100X GDP (Cat. # 30304): 50 µl at 100 mM, use 5 µL of GDP for GDP-labeling of 0.5 mL of cell lysate.
7. HRP-Goat Anti-Rabbit IgG (Cat. # 29002): 50 µL (0.4 µg/mL) in PBS, pH 7.4, contained 50% glycerol.
D. Materials Needed but Not Supplied
1. Stimulated and non-stimulated cell lysates
2. Protease inhibitors
3. 4 °C tube rocker or shaker
4. 0.5 M EDTA at pH 8.0
5. 1.0 M MgCl2
6. 2X reducing SDS-PAGE sample buffer
7. Electrophoresis and immunoblotting systems
8. Immunoblotting wash buffer such as TBST (10 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 0.05%  Tween-20)
9. Immunoblotting blocking buffer (TBST containing 5% Non-fat Dry Milk or 3% BSA)
10. ECL Detection Reagents
E. Example Results
The following figure demonstrates example results seen with the Cdc42 Activation Assay Kit. For reference only.
Cdc42 Activation Assay Kit Cdc42 Activation Assay. MEF cells were treated with (lane 2) or without (lane 1) EGF. Cell lysates were incubated with an anti- Cdc42-GTP monoclonal antibody (Cat. # 26905) (top panel). The precipitated active Cdc42 was immunoblotted with an anti-Cdc42 rabbit polyclonal antibody (Cat # 21010). The bottom panel shows the Western blot with anti-Cdc42 of the cell lysates used (5% of that used in the top panel).

Assay Procedure

A. Reagent Preparation
1X Assay/Lysis Buffer: Mix the 5X Stock (Cat. # 30302) briefly and dilute with deionized water to make 1X buffer. Just prior to usage, add protease inhibitors such as 1 mM PMSF, 10 µg/mL leupeptin, and 10 µg/mL aprotinin.
B. Sample Preparation
Adherent Cells
1. Culture cells (one 10-cm plate, ~107 cells) to approximately 80-90% confluence. Stimulate the cells with activator or inhibitor as desired.
2. Aspirate the culture media and wash twice with ice-cold PBS.
3. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cells (0.5-1 mL per 10 cm tissue culture plate).
4. Place the culture plates on ice for 10-20 minutes.
5. Detach the cells from the plates by scraping with a cell scraper.
6. Transfer the lysates to appropriate size tubes and place on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store the sample (~1-2 mg of total protein) on ice for immediate use, or snap freeze and store at -70°C for future use.
Suspension Cells
1. Culture cells and stimulate with activator or inhibitor as desired.
2. Perform a cell count and then pellet the cells through centrifugation.
3. Aspirate the culture media and wash twice with ice-cold PBS.
4. Completely remove the final PBS wash and add ice-cold 1X Assay/Lysis Buffer (See Reagent Preparation) to the cell pellet (0.5-1 mL per 107 cells).
5. Lyse the cells by repeated pipetting.
6. Transfer the lysates to appropriate size tubes and place them on ice.
7. If nuclear lysis occurs, the cell lysates may become viscous and difficult to pipette. If this occurs, lysates can be passed through a 27½-gauge syringe needle 3-4 times to shear the genomic DNA.
8. Clear the lysates by centrifuging at 12,000 x g and 4°C for 10 minutes.
9. Collect the supernatant and store sample on ice for immediate use, or snap freeze and store at -70°C for future use.
C. In vitro GTPγS/GDP Protein for Positive and Negative controls
Note: In vivo stimulation of cells will activate approximately 10% of the available Cdc42, whereas in vitro GTPγS protein loading will activate nearly 90% of Cdc42.
1. Aliquot 0.5 mL of cell extract (or 1 µg of purified Cdc42 protein) into two microcentrifuge tubes.
2. To each tube, add 20 µL of 0.5 M EDTA (final concentration of 20 mM).
3. Add 5 µL of 100 X GTPγS (Cat. # 30303) to the first tube as a positive control.
4. Add 5 µL of 100 X GDP (Cat. # 30304) to the second tube as a negative control.
5. Incubate both tubes at 30°C for 30 minutes with agitation.
6. Stop loading by placing the tubes on ice and adding 32.5 µL of 1 M MgCl2 (final concentration of 60 mM).
D. Affinity Precipitation of Activated G Protein
1. Aliquot 0.5-1 mL of cell lysates (about 1 mg of total cellular protein) to a microcentrifuge tube.
2. Adjust the volume to 1 mL with 1X Assay/Lysis Buffer (See Reagent Preparation).
3. Add 1 µL anti-Cdc42-GTP antibody (Cat. # 26905).
4. Prepare the protein A/G Agarose bead slurry (Cat. # 30301) by resuspending through vertexing or titrating.
5. Quickly add 20 µL of resuspended bead slurry to above tube.
6. Incubate the tube at 4°C for 1 hour with gentle agitation.
7. Pellet the beads through centrifugation at 5,000 x g for 1 min.
8. Aspirate and discard the supernatant (making sure not to disturb or remove the bead pellet).
9. Wash the beads 3 times with 0.5 mL of 1X Assay/Lysis Buffer, centrifuging and aspirating each time.
10. After the third wash, pellet the beads through centrifugation and carefully remove all the supernatant.
11. Resuspend the bead pellet in 20 µL of 2X reducing SDS- PAGE sample buffer.
12. Boil the sample for 5 minutes.
13. Centrifuge it at 5,000 x g for 10 seconds.
E. Western Blot Analysis
1. Load 15 µL/well of pull-down supernatant to a polyacrylamide gel (17%). It is recommended to include a pre-stained MW standard (as an indicator of a successful transfer in step 3 below).
2. Perform SDS-PAGE following the manufacturer’s instructions.
3. Transfer the gel proteins to a PVDF or nitrocellulose membrane following the manufacturer’s instructions.
Note: Steps 4-11 are at room temperature with agitation
4. Following electroblotting, immerse the PVDF membrane in 100% Methanol for 15 seconds, and then allow it to dry at room temperature for 5 minutes.
Note: If Nitrocellulose is used instead of PVDF, step 4 Should be skipped.
5. Block the membrane with 5% non-fat dry milk or 3% BSA in TBST for 1 hr at room temperature with constant agitation.
6. Wash the blotted membrane three times with TBST, 5 minutes each time.
7. Incubate the membrane with Anti-Cdc42 Rabbit Polyclonal Antibody (Cat. # 21010), which has been freshly diluted 1:50~500 (depending on the amount of Cdc42 proteins in your sample) in 5% non-fat dry milk or 3% BSA in TBST, for 1-2 hr at room temperature with constant agitation or at 4°C overnight.
8. Wash the blotted membrane three times with TBST, 5 minutes each time.
9. Incubate the membrane with a secondary antibody (Cat. # 29002), which is freshly diluted 1:1000 in 5% non-fat dry milk or 3% BSA in TBST, for 1 hr at room temperature with constant agitation.
10. Wash the blotted membrane three times with TBST, 5 minutes each time.
11. Use the detection method of your choice such as ECL.
          Publications          
01. Progesterone receptor potentiates macropinocytosis through CDC42 in pancreatic ductal adenocarcinoma
Oncogenesis. 2024  PMID: 38424455
02. Lung cancer cell-intrinsic IL-15 promotes cell migration and sensitizes murine lung tumors to anti-PD-L1 therapy
Biomark Res. 2024  PMID: 38637902
03. A Natural Small Molecule Mitigates Kidney Fibrosis by Targeting Cdc42‐mediated GSK‐3β/β‐catenin Signaling
Adv Sci (Weinh). 2024  PMID: 38240457
04. Infiltrative vessel co-optive growth pattern induced by IQGAP3 overexpression promotes microvascular invasion in hepatocellular carcinoma
Clin Cancer Res. 2024  PMID: 38470497
05. 25-hydroxycholesterol inhibits human papillomavirus infection in cervical epithelial cells by perturbing cytoskeletal remodeling
J Med Virol. 2023  PMID: 37254637
06. Lithium impacts the function of hematopoietic stem cells via disturbing the endoplasmic reticulum stress and Hsp90 signaling
Food Chem Toxicol. 2023  PMID: 37783420
07. The activated CD36-Src axis promotes lung adenocarcinoma cell proliferation and actin remodeling-involved metastasis in high-fat environment
Cell Death Dis. 2023  PMID: 37612265
08. Glutamine metabolic microenvironment drives M2 macrophage polarization to mediate trastuzumab resistance in HER2‐positive gastric cancer
Cancer Commun (Lond). 2023  PMID: 37434399
09. Cystic fibrosis macrophage function and clinical outcomes after elexacaftor/tezacaftor/ivacaftor
Eur Respir J. 2022  PMID: 36265882
10. ARHGEF37 overexpression promotes extravasation and metastasis of hepatocellular carcinoma via directly activating Cdc42
J Exp Clin Cancer Res. 2022  PMID: 35869555
11. C9orf72 knockdown alleviates hepatic insulin resistance by promoting lipophagy
Biochem Biophys Res Commun. 2022  PMID: 34942529
12. Novel rapid immunohistochemistry using an alternating current electric field identifies Rac and Cdc42 activation in human colon cancer FFPE tissues
Sci Rep. 2022  PMID: 35110666
13. Novel rapid immunohistochemistry using an alternating current electric field identifies Rac and Cdc42 activation in human colon cancer FFPE tissues
Sci Rep. 2022  PMID: 35110666
14. Arhgef6 (alpha-PIX) cytoskeletal regulator signals to GTPases and Cofilin to couple T cell migration speed and persistence
J Leukoc Biol. 2021  PMID: 33527537
15. Glucocorticoid-mediated induction of caveolin-1 disrupts cytoskeletal organization.inhibits cell migration and re-epithelialization of non-healing wounds
Commun Biol. 2021  PMID: 34145387
16. NDRG1 regulates Filopodia-induced Colorectal Cancer invasiveness via modulating CDC42 activity
Int J Biol Sci. 2021  PMID: 33994856
17. Spatiotemporal Control of Actomyosin Contractility by MRCKß Signaling Drives Phagocytosis
bioRxiv,2021  PMID: 36121394
18. Cdc42 Mediates Cancer Cell Chemotaxis in Perineural Invasion
Mol Cancer Res. 2020  PMID: 32086369
19. Mevalonate metabolismûdependent protein geranylgeranylation regulates thymocyte egress
J Exp Med. 2020  PMID: 31722972
20. Progressive Pulmonary Fibrosis Is Caused by Elevated Mechanical Tension on Alveolar Stem Cells
Cell. 2020  PMID: 31866069
21. Spatial distribution of IL4 controls iNKT cell-DC crosstalk in tumors
Cell Mol Immunol. 2020  PMID: 31160756
22. Cdc42 is involved in NC1 peptideûregulated BTB dynamics through actin and microtubule cytoskeletal reorganization
FASEB J. 2019  PMID: 31682474
23. Effects of age-dependent changes in cell size on endothelial cell proliferation and senescence through YAP1
Aging (Albany NY). 2019  PMID: 31487690
24. Inhibition of Cdc42-intersectin interaction by small molecule ZCL367 impedes cancer cell cycle progression.proliferation.migration.and tumor growth
Cancer Biol Ther. 2019  PMID: 30849276
25. Inhibition of Fascin in Cancer and Stromal Cells Blocks Ovarian Cancer Metastasis
Gynecol Oncol. 2019  PMID: 30797592
26. Sertoli cell–specific coxsackievirus and adenovirus receptor regulates cell adhesion and gene transcription via β-catenin inactivation and Cdc42 activation
FASEB J..2019  PMID: 30892947
27. The alteration of RhoA geranylgeranylation and Ras farnesylation breaks the integrity of the bloodûtestis barrier and results in hypospermatogenesis
Cell Death Dis. 2019  PMID:  31171774
28. WiskottûAldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma
Nat Med. 2019  PMID: 30510251
29. Cadmium Activates Noncanonical Wnt Signaling to Impair Hematopoietic Stem Cell Function in Mice
Toxicol Sci.2018  PMID:  29939372
30. Integrating transient cellular and nuclear motions to comprehensively describe cell migration patterns
Sci Rep. 2018  PMID:  29367613
31. MiR-142-3p Inhibits TGF-β3-Induced Blood-Testis Barrier Impairment by Targeting Lethal Giant Larvae Homolog 2
Cell Physiol Biochem. 2018  PMID: 29590647
32. A FAK-YAP-mTOR signaling axis regulates stem cell-based tissue renewal in mice
Cell Stem Cell. 2017  PMID: 28457749
33. Curcumin inhibits growth of human breast cancer cells through demethylation of DLC1 promoter
Mol Cell Biochem..2017  PMID:  27830358
34. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary
PLoS Genet. 2017  PMID: 28072828
35. Heme oxygenase-1 protects spinal cord neurons from hydrogen peroxide-induced apoptosis via suppression of Cdc42/MLK3/MKK7/JNK3 signaling
Apoptosis. 2017  PMID:  27864650
36. High glucose levels promote the proliferation of breast cancer cells through GTPases
Breast Cancer (Dove Med Press). 2017  PMID:  28670141
37. microRNA-206 modulates an Rtn4a/Cxcr4a/Thbs3a axis in newly forming somites to maintain and stabilize the somite boundary formation of zebrafish embryos
Open Biol. 2017  PMID:  28701377
38. Myosin 1g Contributes to CD44 Adhesion Protein and Lipid Rafts Recycling and Controls CD44 Capping and Cell Migration in B Lymphocytes
Front Immunol. 2017  PMID: 29321775
39. Primary angle closure glaucoma (PACG) susceptibility gene PLEKHA7 encodes a novel Rac1/Cdc42 GAP that modulates cell migration and blood-aqueous barrier function
Hum Mol Genet. 2017  PMID:  29016860
40. PTEN controls glandular morphogenesis through a juxtamembrane ß-Arrestin1/ARHGAP21 scaffolding complex
eLife. 2017  PMID: 28749339
41. Mutations of Cystic Fibrosis Transmembrane Conductance Regulator Gene Cause a Monocyte-Selective Adhesion Deficiency
Am J Respir Crit Care Med. 2016  PMID: 26694899
42. Nitric oxide increases the migratory activity of non-small cell lung cancer cells via AKT-mediated integrin αv and β1 upregulation
Cell Oncol (Dordr).2016  PMID: 27376838
43. Opposing effects of actin signaling and LFA-1 on establishing the affinity threshold for inducing effector T-cell responses in mice
Eur J Immunol. 2016  PMID: 27188212
44. Thiopurine Prodrugs Mediate Immunosuppressive Effects by Interfering with Rac1 Protein Function
J Biol Chem. 2016  PMID:  27189938
45. Actin remodeling by Nck regulates endothelial lumen formation
Mol Biol Cell. 2015  PMID: 26157164
46. Axon Targeting of the Alpha 7 Nicotinic Receptor in Developing Hippocampal Neurons by Gprin1 Regulates Growth
J Neurochem. 2014  PMID: 24350810
47. Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation
J Cell Biol. 2014  PMID: 24379416
48. Loss of ARHGDIA expression is associated with poor prognosis in HCC and promotes invasion and metastasis of HCC cells
Int J Oncol. 2014  PMID: 24859471
49. MIF inhibits monocytic movement through a non-canonical receptor and disruption of temporal Rho GTPase activities in U-937 cells
Cytokine.2014  PMID:  25022961
50. Spatial control of Cdc42 signalling by a GM130-RasGRF complex regulates polarity and tumorigenesis
Nat Commun. 2014  PMID: 25208761
51. srGAP3 promotes neurite outgrowth of dorsal root ganglion neurons by inactivating RAC1
Asian Pac J Trop Med. 2014  PMID: 25149377
52. The a4 Nicotinic Receptor Promotes CD4+ T-Cell Proliferation and a Helper T-Cell Immune Response
Mol Pharmacol..2014  PMID:  24107512
53. The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane Recycling in Osteoclasts
J Biol Chem. 2014  PMID: 24821726
54. The tumour suppressor DLC2 ensures mitotic fidelity by coordinating spindle positioning and cellûcell adhesion
Nat Commun. 2014  PMID: 25518808
55. Depletion of the transcriptional coactivators megakaryoblastic leukaemia 1 and 2 abolishes hepatocellular carcinoma xenograft growth by inducing oncogene-induced senescence
EMBO Mol Med. 2013  PMID:  23853104
56. p21-Activated kinase (PAK) regulates cytoskeletal reorganization and directional migration in human neutrophils
PLoS One.2013  PMID:  24019894
57. Epithelial junction formation requires confinement of Cdc42 activity by a novel SH3BP1 complex
J Cell Biol. 2012  PMID: 22891260
58. Essential Role for Rac1 in Cocaine-Induced Structural Plasticity of Nucleus Accumbens Neurons
Nat Neurosci. 2012  PMID: 22522400
59. Invadopodia and rolling-type motility are specific features of highly invasive p190(bcr-abl) leukemic cells
Eur J Cell Biol.2012  PMID:  22717125
60. ProBDNF Collapses Neurite Outgrowth of Primary Neurons by Activating RhoA
PLoS One. 2012  PMID: 22558255
61. Enterohemorrhagic Escherichia coli infection stimulates Shiga toxin 1 macropinocytosis and transcytosis across intestinal epithelial cells
Am J Physiol Cell Physiol. 2011  PMID: 21832249
62. Polarized migration of lymphatic endothelial cells is critically dependent on podoplanin regulation of Cdc42
Am J Physiol Lung Cell Mol Physiol. 2011  PMID: 21036919
63. The Aarskog-Scott Syndrome Protein Fgd1 Regulates Podosome Formation and Extracellular Matrix Remodeling in Transforming Growth Factor á-Stimulated Aortic Endothelial Cells
Mol Cell Biol. 2011  PMID: 21911474