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

1. Membrane strips coated with various lipids are incubated in 10 ml of blocking buffer for 1 h at room temperature in dark (the commercially available membrane strip has been coated with lipids).
   
2. 20-60 μg of proteins are added to 6 ml incubation buffer with membrane strips and incubated overnight at 4 °C. In our study, we used Flag-tagged protein purified from 293T cells (Zhang et al., 2012). However, proteins purified from other sources can all be used. Negative controls should be included according to specific proteins used in this assay.
   
3. Membrane strips are washed extensively with wash buffer for 3 times at RT, 10 min each wash on rotator.
   
4. Membrane strips with bounded proteins are incubated with primary antibodies (Anti-Flag M2 from mouse, 1:1,000) in incubation buffer for 3 h at RT.
   
5. Wash 3 times at RT with wash buffer, 10 min each wash on rotator.
   
6. Incubate membrane strips with HRP-conjugated secondary antibodies (Goat anti-Mouse LgG (H+L) HRP, 1:10,000) in incubation buffer for 1 h at RT.
   
7. Membranes are washed as above and protein-antibody interaction is detected using SuperSignal West Pico reagent.
   

Recipes

1. Blocking buffer
   25 mM Tris-Cl
   150 mM NaCl
   0.1% Tween-20
   1% non-fat milk
   
2. Incubation buffer
   25 mM Tris-Cl
   150 mM NaCl
   0.1% Tween-20
   1% non-fat milk
   2 mM Ca²⁺ chloride
   1 mM Zn²⁺ sulfate
   
3. Wash buffer
   25 mM Tris-Cl
   150 mM NaCl
   0.1% Tween-20
   2 mM Ca²⁺ chloride
   1 mM Zn²⁺ sulfate
   

Acknowledgments

This protocol is adapted from Zhang et al. (2012) and Wang et al. (2010).

References

1. Wang, X., Li, W., Zhao, D., Liu, B., Shi, Y., Chen, B., Yang, H., Guo, P., Geng, X., Shang, Z., Peden, E., Kage-Nakadai, E., Mitani, S. and Xue, D. (2010). Caenorhabditis elegans transthyretin-like protein TTR-52 mediates recognition of apoptotic cells by the CED-1 phagocyte receptor. Nat Cell Biol 12(7): 655-664.
   
2. Zhang, Y., Wang, H., Kage-Nakadai, E., Mitani, S. and Wang, X. (2012). C. elegans secreted lipid-binding protein NRF-5 mediates PS appearance on phagocytes for cell corpse engulfment. Curr Biol 22(14): 1276-1284.