Lipid Beads

Echelon’s Lipid Strips and Arrays are simple, but highly effective and widely used assays for determining lipid-protein interactions. Here we provide an extensive protocol as a guide and reference for use with your protein of interest. The protocol includes suggestions for positive controls and buffers as well as general recommendations for a successful assay.

Protocol

Lipid Beads^TM are designed for use in protein pull-down experiments. Possible uses are to test for binding in a solution of purified protein, in a cell lysate, or to test binding of radiolabeled in vitro translation products.

1. Pellet beads by centrifugation at 1,000 x g or lower.
2. Remove the supernatant and resuspend the beads in an equal volume of wash-binding buffer. Add 1-10 μg of protein, diluted in binding buffer, to 50 – 100 μL of beads.
   -When using cell lysate, avoid harsh lysis conditions which may interfere with lipid structure and binding.
3. Incubate the protein-bead solution for 1-3 hours. The incubation can be done at room temperature or at 4° C, depending on the stability of your protein. Continuous motion is required to keep beads in suspension.
4. Pellet the beads and remove the supernatant.
   -Carefully remove the wash solution to avoid losing beads.
5. Wash the beads with 10X excess of the wash/binding buffer.
6. Repeat wash steps 4 and 5, two to five times.
7. Following the last wash, elute bound proteins by adding an equal volume of 2X Laemmli sample buffer (or similar) to beads and heat to 95 °C for 5-10 minutes.
8. Following heating, pellet the beads, remove the supernatant, and store  supernatant at 4°C until analysis.

Proteins can be separated by SDS-PAGE and analyzed by Coomassie blue staining of the gel, protein transfer and immunoblotting to detect proteins of interest, or autoradiography. Additional considerations for identification of lipid binding proteins by mass spectrometry can be found here.

Buffers

We recommend 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.25% Igepal as a starting point for the wash and binding buffer. If the protein is sticking nonspecifically to Control Beads, the detergent concentration can be raised to 0.5% or higher. Other possible buffers are listed below.

Notes

1. Lipid Coated Beads are available in 1 mL and 0.1 mL sizes and come as a 50% slurry in 1X PBS Buffer.
2. An order of 1 mL comes with 200 µL of Control Beads. Control Beads are also available for purchase and their use is suggested for analysis.
3. Store at 2-8°C. Product is temperature and light sensitive. Do not freeze.
4. Centrifuge the beads at 1,000 x g or lower. Do not centrifuge the beads at high speed as this can crush the beads.
5. There are a total of 10 nmoles of bound lipid per 1 mL of beads.
6. The beads range from 45 – 165 micrometers in diameter.
7. Use 50-100 μL of beads for each pull-down assay. (Note: 50 μL of bead = 100 μL of 50% slurry)

References

General

1. Hawse WF, Cattley RT. T cells transduce T-cell receptor signal strength by generating different phosphatidylinositols. J Biol Chem. 2019;294(13):4793-805.
2. Fang C, Manes TD, Liu L, Liu K, Qin L, Li G, et al. ZFYVE21 is a complement-induced Rab5 effector that activates non-canonical NF-κB via phosphoinosotide remodeling of endosomes. Nature Communications. 2019;10(1):2247.

Mass Spec Identification

1. Kattan, R et al. (2022) Interactome analysis of human phospholipase D and phosphatidic acid-associated protein network. Molecular & Cellular Proteomics 21 (2)
2. Durrant, T et al. (2019) Identification of PtdIns(3,4)P₂ effectors in human platelets using quantitative proteomics. BBA – Molecular and Cell Biology of Lipids, 1865.
3. Zhu, Z et al. (2019) Ceramide regulates interaction of Hsd17b4 with Pex5 and function of peroxisomes. Biochimica et Biophysica Acta (BBA) – Molecular and Cell Biology of Lipids.