Product Uses Include
- Arf6 signaling pathway studies
- Arf6 activation assays with primary cells
- Studies of Arf6 activators and inactivators
- Arf6 activation assays with limited material
- High throughput screens for Arf6 activation
Introduction
With the new Arf6 G-LISA® kit you can now measure Arf6 activation from cell and tissue samples in less than 3 h. G-LISA® requires only 1-5% of the material needed for a conventional pull-down assay. You will also be able to handle large sample numbers and generate quantitative results. For a more detailed introduction on G-LISA® assays and a listing of other available G-LISA® kits, see our main G-LISA page.
The Arf6 G-LISA® kit contains an Arf-GTP-binding protein linked to the wells of a 96 well plate. Active, GTP-bound Arf6 in cell/tissue lysates will bind to the wells while inactive GDP-bound Arf6 is removed during washing steps. The bound active Arf6 is detected with a Arf6 specific antibody. The degree of Arf6 activation is determined by comparing readings from activated cell lysates versus non-activated cell lysates.
Kit contents
The kit contains sufficient reagents to perform 96 Arf6 activation assays. Since the Arf-GTP affinity wells are supplied as strips and the strips can be broken into smaller pieces, each kit can be used for anywhere from one to multiple assays. The following components are included in the kit:
- 96 well Arf6-GTP binding plate (divisible into 12 strips of 8 wells each)
- Anti-Arf6 antibody
- HRP-labeled secondary antibody
- Arf6 control protein (constitutively active Arf6)
- Cell Lysis buffer
- Wash buffer
- Binding buffer
- Antigen presenting buffer
- Antibody dilution buffer
- HRP detection reagent A
- HRP detection reagent B
- HRP stop solution
- Precision Red™ advanced protein assay reagent (Cat. # ADV02)
- Protease inhibitor cocktail (Cat. # PIC02)
- Manual with detailed protocols and extensive troubleshooting guide
Equipment needed
- 96-well plate spectrophotometer capable of reading 490 nm wavelength
- Multichannel or multidispensing pipettor
- Orbital microplate shaker capable of at least 200 rpm shaking (400 rpm is optimal)
Example results
Figure 1. Arf6 activation measured by G-LISA. Lysates for Arf6 G-LISA were prepared from MDCK cells that were either attached to tissue culture plates (Activated) or kept in suspension for 2 hours (Control). 12.5, 6.25, and 3.1 µg of cell lysates were subjected to the G-LISA assay. Absorbance was read at 490 nm. Data are background subtracted.
Go to main G-LISA™ page
For product Datasheets and MSDSs please click on the PDF links below.
G-LISA Activation Assay Technical Guide download here
Jackson, M. R. et al. Heterozygous loss of function of IQSEC2/Iqsec2 leads to increased activated Arf6 and severe neurocognitive seizure phenotype in females. Life Sci. Alliance 2, (2019).
Rafiq, N. B. M. et al. Podosome assembly is controlled by the GTPase ARF1 and its nucleotide exchange factor ARNO. J. Cell Biol. 216, 181–197 (2017).
Stepicheva, N. A., Dumas, M., Kobi, P., Donaldson, J. G. & Song, J. L. The small GTPase Arf6 regulates sea urchin morphogenesis. Differentiation 95, 31–43 (2017).
Biesemann, A., Gorontzi, A., Barr, F. & Gerke, V. Rab35 protein regulates evoked exocytosis of endothelial Weibel–Palade bodies. J. Biol. Chem. 292, 11631–11640 (2017).
Andrews, A. M., Lutton, E. M., Merkel, S. F., Razmpour, R. & Ramirez, S. H. Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury. Front. Cell. Neurosci. 10, 43 (2016).
Rennoll-Bankert, K. E. et al. RalF-mediated activation of Arf6 controls Rickettsia typhi invasion by co-opting phosphoinositol metabolism. Infect. Immun. 84, 3496–3506 (2016).
Coming soon! If you have any questions concerning this product, please contact our Technical Service department at tservice@cytoskeleton.com
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