Search

In vitro Lipid Binding Assay   

Download PDF How to cite Favorites Q&A Share your feedback

In this protocol

Original research article

A brief version of this protocol appeared in:
Current Biology
Jul 2012

Abstract

This is a protocol to examine in vitro protein-lipid binding using membrane strips coated with various lipids. It has been successfully used to study in vitro interaction between lipids and C. elegans proteins.

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.
Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Zhang, Y. and Wang, X. (2013). In vitro Lipid Binding Assay. Bio-protocol 3(9): e694. DOI: 10.21769/BioProtoc.694.
Q&A

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data including images for the troubleshooting.

You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.