Separation of membrane proteins according to their hydropathy by serial phase partitioning with Triton X-114

The detergent Triton X-114, because of its convenient cloud point temperature (22 °C), has been used extensively to extract membrane proteins and to separate them in two phases according to their hydropathy. The upper detergent-poor phase contains mostly hydrophilic proteins, whereas hydrophobic ones are found mainly in the lower detergent-rich phase. In this work, we developed a method to fractionate membrane proteins and estimate their hydropathy based on a series of cloud point partitions with Triton X-114. With this method, beetroot plasma membrane proteins were separated in different fractions according to their hydropathy, following the binomial distribution law as expected. This method revealed the presence of both hydrophilic and hydrophobic Ca²⁺-dependent protein kinases in those membranes. At least five distinct Ca²⁺-dependent kinases were observed in in-gel kinase activity assays. This separation procedure was also used as the first step in the purification of a hydrophobic 60-kDa kinase.