Choosing Your Bilayer Composition
The first step when setting out to make bilayers is to decide on the lipid composition. Depending on your application, your lipid composition will vary, but most bilayers will primarily contain phosphatidylcholine (PC), which provides the structural framework. You can use a polyethylene glycol (PEG) cushion to improve the uniformity of your bilayer and reduce protein aggregation. To do so, include a small fraction of lipids with PEG5,000 polymers covalently attached to the lipid headgroups. However, be aware that too much PEG will compromise the fluidity of your bilayer. You can use different strategies to attach proteins to the bilayers; the two most common are via Ni-NTA lipids (which bind His-tagged proteins) and biotinylated lipids (which bind streptavidin, which in turn captures proteins tagged with biotin).Creating the Bilayers – it’s a Kind of Magic
Below is a table that shows an example of a typical lipid mixture containing DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) as the main structural component, DGS-NTA (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl] (ammonium salt)) for binding His-tagged proteins, DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine)-biotin for attaching streptavidin, and PEG5,000-DOPE to improve the uniformity of the bilayer. The lipids are added at specific molar percentages. From the lipid concentrations and molecular weights, you can calculate the required volumes to get a rehydrated lipid mix of the desired final concentration (for example 1 mg/ml).Lipid Component | Mol % |
DOPC | 96.5 |
DGS-NTA | 2 |
DOPE-biotin | 1 |
PEG 5,000-DOPE | 0.5 |
Total | 100 |
Are Your Bilayers Mobile?
It’s important to have mobile bilayers so that your lipids and proteins can diffuse in the same way as they would in an actual cell membrane. To check the mobility of your bilayers, use a microscopy technique called Fluorescence Recovery After Photobleaching (FRAP)2, which relies on the diffusion of fluorescently-labeled molecules into a photobleached sample area.. You should check both the mobility of your lipids (by using fluorescently-tagged lipids, e.g. rhodamine-tagged lipids) and the mobility of your attached proteins (by using fluorescent streptavidin or fluorescently-labelled proteins).You’re in Business…
In summary, bilayers are a cool tool for studying cell-cell interactions or membrane protein dynamics in a reductionist system that you can control. My work with bilayers has helped me show how T cells are able to transmit an activating signal from the outside of the cell to the intracellular signaling pathway. Maybe bilayers could help you too!References
- Beemiller P et. al. 2012. Imaging and analysis of OT1 cell activation on lipid bilayers. Protoc. Exch. doi:10.1038/protex.2012.028
- Axelrod D et.al. 1976. Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16: 1055–69.