In vitro Lipid Binding Assay

Materials and Reagents

1. Membrane Lipid strip (Echelon Bioscience, catalog number: P-6001 )
   
2. Proteins of interest (EGFP-NRF-5-Flag, EGFP-Flag as control)
   
3. Anti-Flag M2 antibody (Sigma-Aldrich, catalog number: A2220 )
   
4. Primary antibodies (Anti-Flag M2 from Mouse, sigma, catalog number: F1804 )
   
5. HRP-conjugated secondary antibodies (e.g. Goat anti-Mouse LgG (H+L) HRP, The Jackson Laboratory, catalog number: 115-035-003 )
   
6. SuperSignal West Pico reagent (Thermo Fisher Scientific, catalog number: 34080 )
7. 293T cells
   
8. Tris-Cl
   
9. NaCl
   
10. Tween-20
    
11. Ca²⁺ chloride
    
12. Zn²⁺ sulfate
    
13. Non-fat milk
    
14. Blocking buffer (see Recipes)
    
15. Incubation buffer (see Recipes)
    
16. Wash buffer (see Recipes)
    

Equipment

1. Vortexer
   
2. Rotator
   

Procedure