Bicelle Preparation

1, 2-Dihexanoyl-sn-Glycero-3-Phosphocholine


An effective and convenient method for preparing bicelles makes use of a buffer solution containing 10mM phosphate buffer, pH 6.6, 0.15 mM sodium azide, 93% H2O (HPLC-grade), 7% D2O (99.9%). Below, this solution will simply be referred to as buffer.


DMPC/DHPC stock solutions containing a total of 15% w/v (150mg lipid/ml) are prepared as follows:

  1. Add buffer to the lyophilized lipid mixture. 50 mg lipid mixture, 280 mg buffer 200 mg lipid mixture, 1130 mg buffer
  2. Let the mixtures hydrate at room temperature (18-220C) for several hours. Lipid mixtures with a “q” of 2.8 – 3.0, the hydration is complete in 2 – 3 hours. Lipid mixtures with a “q” of 3.25 – 3.5 require 24 hours for complete hydration. Accelerated hydration (one hour) may be effected by heating any mixture to 40°C for 10 minutes and cycling to 18°C twice, then briefly vortexing.


Two volumes of protein solution are added to one volume of bicelle solution.



Tjandra & Bax (1) recently developed a new nuclear magnetic resonance (NMR) technique that gently aligns protein molecules in a bath of liquid crystals, allowing researchers to determine how each bond between neighboring atoms is oriented with respect to the rest of the molecule. By compiling all such orientations between atoms, a precise map of the protein can be derived. In aqueous solution, just above room temperature, the lipids switch from a gel to a Liquid Crystal (LC) phase, where they form discshaped particles, often referred to as bicelles (2), with diameters of several hundred angstroms and thicknesses of ~40Å. The lipids are diamagnetic, and, as a result, the bicelles orient with their normal orthogonal to the magnetic field. However, the lifetimes and temperature ranges of orientation for these samples are critically dependent on sample composition and experimental conditions. Losonczi & Prestegard (3) demonstrated that doping dilute bicelle solutions with small amounts of charged amphiphiles substantially improves the stability and degree of alignment, as well as extends the temperature range of orientation for these systems.

An explanation of the dependence of bicelle aggregation on sample composition is proposed based on the DLVO theory of colloids. Crowell & Macdonald (4) used solidstate phosphorus (31P) and deuterium (2H) NMR spectroscopy over the temperature range of 25-50°C to investigate bilayered micelles (bicelles) composed of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) and 1,2-Dihexanoyl-sn-Glycero-3-Phosphocholine (DHPC) in the presence of either the anionic lipid 1,2-Dimyristoyl-sn-Glycero-3-PhosphoGlycerol (DMPG) or the cationic lipid 1,2-Dimyristoyl-3-TrimethylAmmoniumPropane (DMTAP). The 31P-NMR spectra demonstrate that bicellar structures form with DMPG/DMPC ratios ranging from 0 to 50/50 and with DMTAP/DMPC ratios from 0 to 40/60, while the overall concentration of DHPC remains constant. The formation of bicelles containing charged amphiphiles is contingent upon the presence of NaCl, with 50 mM NaCl being sufficient for bicelle formation at all concentrations of charged amphiphile investigated, while 150 mM NaCl affords better resolution of the various 31PNMR resonance signals. The 2H-NMR spectra demonstrate that the quadrupolar splittings Dv of headgroup deuterated DMPC change inversely as a function of the amount of negative versus positive charge present, and that the changes for deuterons on the α-carbon are opposite in sense to those for deuterons on the ß-carbon. This indicates that headgroup deuterated phosphatidylcholine functions as a molecular voltmeter in bicelles in much the same fashion as it does in spherical vesicles.


In order to increase the stability of bicelles over a wide pH range Ottiger & Bax (5) demonstrated that mixtures of DitetradecylPhosphatidylcholine (14-O-PC) or DidodecylPhosphatidylcholine (12-O-PC) and DihexylPhosphatidylcholine (6-O-PC) in water form lyotropic liquid crystalline phases under similar conditions as previously reported for bicelles consisting of DMPC and DHPC. The carboxyester bonds present in DMPC and DHPC are replaced by ether linkages in their alkyl analogs, which prevents acid or basecatalyzed hydrolysis of these compounds. 15N-1H dipolar couplings measured for ubiquitin over the 2.3-10.4 pH range indicate that this protein retains a backbone conformation which is very similar to its structure at pH 6.5 over this entire range. Also, Cavagnero et al. (6) prepared and characterized a novel bicelle system composed of 1,2-Di-O-Dodecyl-sn-Glycero-3-Phosphocholine (DIODPC) and 3-(ChlorAmidoPropyl)-dimethylammonio-2-Hydroxyl-1-Propane Sulfonate (CHAPSO). At the optimal DIODPC/CHAPSO molar ratio of 4.3:1, this medium becomes magnetically oriented from pH 6.5 down to pH 1.0. Unlike previously reported acyl phospholipid bicelle preparations, these bicelles are chemically stable at low pH and are capable of inducing protein alignment, as illustrated by the large residual dipolar couplings measured for rusticyanin from Thiobacillus ferrooxidans at pH 2.1. The DIODPC/CHAPSO system is particularly useful for measuring residual dipolar couplings of macromolecules that require very acidic conditions.


Struppe et al. (7) studied the deuterium NMR spectra of Myr-d27-GNAAAAKKGSEQES (Cat14), the N-terminal 14-residue peptide from the catalytic subunit of cAMP-dependent protein kinase A (PKA), to illustrate how magnetically aligned neutral and acidic phospholipid bicelles can be used to characterize the ordering and mode of binding of peptides to membranes. Since Cat14 is electrically neutral, the major interaction responsible for binding is the insertion of the myristoyl group into the hydrophobic core of the bilayer. The inclusion of 25% Phosphatidylserine (DMPS) or Phosphatidylglycerol (DMPG) into Phosphatidylcholine DMPC:DHPC bicelles results in a moderate increase in the ordering of the peptide relative to the bicelle normal, presumably because of favorable electrostatic interactions between the phospholipid headgroups and the two lysines in positions 7 and 8. Successful preparation of acidic bicelles was achieved by careful adjustment of lipid composition, pH and ionic strength. Prosser et al. (8) developed a new phospholipid chelate complexed with ytterbium (DMPE-DTPA:Yb3+) that is shown to be readily incorporated into a model membrane system, which may then be aligned in a magnetic field such that the average bilayer normal lies along the field. This so-called positively ordered smectic phase, whose lipids consist of less than 1% DMPE-DTPA:Yb3+, is ideally suited to structural studies of membrane proteins by solid-state NMR, low-angle diffraction, and spectroscopic techniques that require oriented samples. The chelate, DMPE-DTPA:Yb3+, which strongly binds the lanthanide ions and serves to orient the membrane in a magnetic field, prevents direct lanthanide-protein interactions and significantly reduces paramagnetic shifts and line broadening. The greatest advantage of the positively aligned lanthanide-chelate membranes lies in their application to the study of large immobile membrane proteins. Similar low-spin lanthanide chelates may have applications in field-ordered solution NMR studies of water soluble proteins and in the design of new magnetically aligned liquid crystalline phases.









NOTE: DHPC is extremely hygroscopic; prepare solutions in dry box or dilute with buffer immediately after opening.



  1. Maceachern, L., A. Sylvester, A. Flynn, A. Rahmani, and M.R. Morrow. (2013). Dependence of Bicellar System Phase Behavior and Dynamics on Anionic Lipid Concentration. Langmuir [PubMed]
  2. Durr, U.H., M. Gildenberg, and A. Ramamoorthy. (2012). The Magic of Bicelles Lights Up Membrane Protein Structure. Chem Rev [PubMed]
  3. Ellena JF, Burnitz MC, Cafiso DS.(2003) Location of the myristoylated alanine-rich C-kinase substrate (MARCKS) effector domain in negatively charged phospholipid bicelles. Biophys J. 2003 Oct; 85(4): 2442-8. [PubMed]
  4. Crowell KJ; Macdonald PM, (1999) Surface charge response of the phosphatidylcholine head group in bilayered micelles from phosphorus and deuterium nuclear magnetic resonance. Biochim Biophys Acta, 1416:12, 2130 
  5. Ottiger M; Bax A, (1999) Bicelle-based liquid crystals for NMRmeasurement of dipolar couplings at acidic and basic pH values. J Biomol NMR, 13:2, 18791 
  6. Cavagnero S; Dyson HJ; Wright PE, (1999) Improved low pH bicelle system for orienting macromolecules over a wide temperature range. J Biomol NMR, 13:4, 38791 
  7. Prosser RS; Volkov VB; and Shiyanovskaya IV, (1998) Novel ChelateInduced Magnetic Alignment of Biological Membranes. Biophys J. 75: 21632169 
  8. Struppe J; Komives EA; Taylor SS; Vold RR, (1998) 2H NMR studies of a myristoylated peptide in neutral and acidic phospholipid bicelles. Biochemistry, 37:44, 155237 
  9. Losonczi JA; Prestegard JH, (1998) Improved dilute bicelle solutions for highresolution NMR of biological macromolecules. J Biomol NMR,12:3, 44751 
  10. Tjandra, N; Bax, A, (1997) Direct Measurement of Distances and Angles in Biomolecules by NMR in a Dilute Liquid Crystalline Medium. Science, 278, 11113. 
  11. Sanders CR II; Schwonek JP, (1992) Characterization of magnetically orientable bilayers in mixtures of dihexanoylphosphatidylcholine and dimyristoylphosphatidylcholine by solidstate NMR. Biochemistry, 31:37, 8898905. 

Current Applications of Bicelle Preparation

  • Ikeda, A., K. Kiguchi, T. Hida, K. Yasuhara, K. Nobusawa, M. Akiyama, and W. Shinoda. (2014). [70]fullerenes assist the formation of phospholipid bicelles at low lipid concentrations. Langmuir 30:12315-20. [PubMed]
  • Beaugrand, M., A.A. Arnold, J. Henin, D.E. Warschawski, P.T. Williamson, and I. Marcotte. (2014). Lipid Concentration and Molar Ratio Boundaries for the Use of Isotropic Bicelles. Langmuir. [PubMed]
  • Cook, G.A., L.A. Dawson, Y. Tian, and S.J. Opella. (2013). The Three Dimensional Structure and Interaction Studies of HCV p7 in DHPC by Solution NMR. Biochemistry. [PubMed]
  • Brindley AJ, Martin RW. (2012, May 8). Effect of Divalent Cations on DMPC/DHPC Bicelle Formation and Alignment. Langmuir. [Epub ahead of print] [Abstract]
  • Gelen Rodríguez, Laia Rubio, Mercedes Cócera, Joan Estelrich, Ramon Pons, Alfonso de la Maza, Olga López. Application of Bicellar Systems on Skin: Diffusion and Molecular Organization Effects. Langmuir, 2010, 26:10578–10584. [Abstract]