Product Uses Include
The principle of the assay is shown schematically below (Figure 1). The assay is based upon the fact that a Rho family effector protein is known to bind preferentially to the active (GTP-bound) form of its target GTPase (1). In the case of RhoA activation, the rhotekin-RBD effector domain is used to make the affinity beads (2). In the case of Cdc42 and Rac1 activation, the PAK-PBD effector domain is used for affinity beads (3). Example Western blot results and detailed protocols can be viewed by clicking the documents tab above and downloading the pdf datasheet.
The kit contains sufficient materials for 10 assays each for RhoA, Rac1, and Cdc42 (depending on activation levels in cells), including reagents for positive and negative controls. The following components are included:
Please check out the new versions of Activation Assays and associated products:
G-LISA® Activation Assays:
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)
Large Pull-down Activation Assays:
Cdc42 Activation Assay Biochem Kit, bead pull-down format (Cat.# BK034)
Rac1 Activation Assay Biochem Kit, bead pull-down format (Cat.# BK035)
RhoA Activation Assay Biochem Kit, bead pull-down format (Cat.# BK036)
Anti-Cdc42 monoclonal antibody (Cat.# ACD03)
Anti-Rac1 monoclonal antibody (Cat.# ARC03)
Anti-RhoA monoclonal antibody (Cat.# ARH03)
GST-tagged Rhotekin-RBD protein on colored agarose beads (Cat. # RT02)
GST-tagged PAK-PBD protein on colored agarose beads (Cat. # PAK02)
His-tagged RhoA protein (Cat. # RH01)
His-tagged Rac1 protein (Cat. # RC01)
His-tagged Cdc42 protein (Cat. # CD01)
|Flores-Muñoz, Carolina et al.||The Long-Term Pannexin 1 Ablation Produces Structural and Functional Modifications in Hippocampal Neurons||Cells||2022||ISSN 2073-4409|
|Moreiras, Hugo et al.||Melanocore uptake by keratinocytes occurs through phagocytosis and involves protease-activated receptor-2 internalization||Traffic||2022||ISSN 1600--0854|
|Liu, Yao et al.||Exosomes derived from stem cells from apical papilla promote craniofacial soft tissue regeneration by enhancing Cdc42-mediated vascularization||Stem Cell Research and Therapy||2021||ISSN 1757-6512|
|Bian, Rui et al.||Research paper rac gtpase activating protein 1 promotes gallbladder cancer via binding DNA ligase 3 to reduce apoptosis||International Journal of Biological Sciences||2021||ISSN 1449-2288|
|Qi, Yan et al.||Rhoa/rock pathway activation is regulated by at1 receptor and participates in smooth muscle migration and dedifferentiation via promoting actin cytoskeleton polymerization||International Journal of Molecular Sciences||2020||ISSN 1422-0067|
|Frye, Maike et al.||Ephrinb2-ephb4 signalling provides rho-mediated homeostatic control of lymphatic endothelial cell junction integrity||eLife||2020||ISSN 2050-084X|
|Brisson, Lucie et al.||P2X7 receptor promotes mouse mammary cancer cell invasiveness and tumour progression, and is a target for anticancer treatment||Cancers||2020||ISSN 2072-6694|
|Dupraz, Sebastian et al.||RhoA Controls Axon Extension Independent of Specification in the Developing Brain||Current Biology||2019||ISSN 0960-9822|
|Birkl, Dorothee et al.||TNFα promotes mucosal wound repair through enhanced platelet activating factor receptor signaling in the epithelium||Mucosal Immunology||2019||ISSN 1935-3456|
|Tang, Lian et al.||RhoA/ROCK signaling regulates smooth muscle phenotypic modulation and vascular remodeling via the JNK pathway and vimentin cytoskeleton||Pharmacological Research||2018||ISSN 1096-1186|
|Cheng, Lili et al.||Zoledronate dysregulates fatty acid metabolism in renal tubular epithelial cells to induce nephrotoxicity||Archives of Toxicology||2018||ISSN 1432-0738|
|Tiwari, Richa et al.||Depletion of keratin 8/18 modulates oncogenic potential by governing multiple signaling pathways||FEBS Journal||2018||ISSN 1742-4658|
|Pan, Yu et al.||Dissection of glomerular transcriptional profile in patients with diabetic nephropathy: SRGAP2a protects podocyte structure and function||Diabetes||2018||ISSN 1939-327X|
|Li, Yan et al.||Aerobic exercise regulates Rho/cofilin pathways to rescue synaptic loss in aged rats||PLoS ONE||2017||ISSN 1932-6203|
|Majumder, Piyali et al.||Cellular levels of Grb2 and cytoskeleton stability are correlated in a neurodegenerative scenario||DMM Disease Models and Mechanisms||2017||ISSN 1754-8411|
|Bon, Emeline et al.||SCN4B acts as a metastasis-suppressor gene preventing hyperactivation of cell migration in breast cancer||Nature Communications||2016||ISSN 2041-1723|
|Zhang, Liang et al.||A lateral signalling pathway coordinates shape volatility during cell migration||Nature Communications||2016||ISSN 2041-1723|
|Liu, Chunqiao et al.||A secreted WNT-ligand-binding domain of FZD5 generated by a frameshift mutation causes autosomal dominant coloboma||Human Molecular Genetics||2016||ISSN 1460-2083|
|Medapati, Manoj Reddy et al.||RAGE mediates the pro-migratory response of extracellular S100A4 in human thyroid cancer cells||Thyroid||2015||ISSN 1557-9077|
|Tang, Jianjun et al.||Paradoxical role of CBX8 in proliferation and metastasis of colorectal cancer||Oncotarget||2014||ISSN 1949-2553|
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: The beads conjugated to the respective effector protein that recognizes the active form of each GTPase will bind to the GDP-bound GTPase with a much lower affinity than the GTP-bound GTPase. If too many beads are added to the pull-down assay there will be significant binding to inactive (GDP-bound) GTPases. The result of this will be an underestimation of GTPase activation. For this reason, we highly recommend performing a bead titration to determine optimal conditions for any given GTPase activation or inactivation assay. Once optimal conditions have been established, bead titrations should no longer be necessary.
Question 3: How can I test whether the beads are working properly?
Answer 3: A standard biological assay for the beads consists of a GTPase protein pull-down from cells loaded with either GTPγS (Cat. # BS01) or GDP. Here are guidelines to follow (see Cat. # BK030 manual 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 GTPase with the nonhydrolysable GTP analog (GTPγS). This is an excellent substrate for the 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 GTPase protein will load with non-hydrolysable GTPγS and will be “pulled-down” with the 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 GTPase with GDP will inactivate the GTPase and this complex will bind very poorly to the beads.
If you have any questions concerning this product, please contact our Technical Service department at email@example.com