The resulting two-dimensional gel electrophoresis pattern (Fig. ), has reduced horizontal streaking and greater spot resolution compared to the gel pattern from acetone:methanol precipitation (Fig. ). Tri-n-butylphosphate, a lipophilic solvent, is thought to extract lipids by forming micelles (8) and may improve the efficiency of acetone:methanol delipidation while retaining the acetone:methanol protein precipitation benefits of concentrating and desalting proteins. A more complete delipidation may reduce the intensity of the precipitate's hydrophobic interaction with aqueous buffers favoring more complete protein solubilization in aqueous buffers and thus greater two-dimensional gel electrophoresis spot resolution. Due to its simplicity this delipidation and protein purification technique is easily applicable to other lipid-dense samples intended for isoelectric focusing and two-dimensional gel electrophoresis. Because this method requires minimal handling, is short in duration, is inexpensive, and does not require specialised equipment, it becomes useful for high-throughput approaches.
In summary, the addition of tri-n-butylphosphate to acetone:methanol provides a simple and effective chemical delipidation technique that results in precipitated protein that is efficiently soluble in electrophoresis buffers. Compared to samples treated with acetone:methanol (8: 1), samples treated with the tri-n-butylphosphate:acetone:methanol (1:12:1) result in the two-dimensional gel electrophoresis patterns with less horizontal streaking and greater spot resolution. This method is suitable for high-throughput approaches and can easily be adapted to other lipid-rich biological samples requiring protein purification prior to two-dimensional gel electrophoresis.