Biochemical analysis of membrane proteins from an early maturation stage of phagosomes

We used an improved technique for pulse-chase labeling of phagosomes using custom-made magnetic microparticles. With the help of a permanent magnet we purified both newly formed, nascent and early matured (i.e., 5-min-old) co ndensed phagosomes in high amounts. The protein patterns of membrane protei ns of newly formed phagosomes and 5-min-old condensed ones were compared by two-dimensional (2-D) electrophoresis. The protein patterns allowed the de tection of protein spots that changed in abundance between these two stages . Three protein spots abundant in condensed phagosomes only and one spot we ll-stained in both stages were collected from ten preparative Coomassie bri lliant blue-stained 2-D gels. Following microdigestion, selected purified o ligopeptides were sequenced by Edman degradation. While the oligopeptide se quences of proteins from two spots showed high homology to an already seque nced 25 kDa calcium binding protein, the other two showed no significant ho mology to protein sequences available in sequence databases. Presently poly merase chain reaction (PCR) and cloning experiments are set up to reveal th e cDNAs of these proteins in order to study their function by knock-out and gene replacement experiments.