Arf6 G-LISA Activation Assay Kit (colorimetric)

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:

1. 96 well Arf6-GTP binding plate (divisible into 12 strips of 8 wells each)
2. Anti-Arf6 antibody
3. HRP-labeled secondary antibody
4. Arf6 control protein (constitutively active Arf6)
5. Cell Lysis buffer
6. Wash buffer
7. Binding buffer
8. Antigen presenting buffer
9. Antibody dilution buffer
10. HRP detection reagent A
11. HRP detection reagent B
12. HRP stop solution
13. Precision Red advanced protein assay reagent (Cat. # ADV02)
14. Protease inhibitor cocktail (Cat. # PIC02)
15. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

1. 96-well plate spectrophotometer capable of reading 490 nm wavelength
2. Multichannel or multidispensing pipettor
3. 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 ug 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

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