Isolation and Western Blotting of Latex-Bead Phagosomes to Track Phagosome Maturation.
Phagocytosis plays an essential role in the immune system for the defense against invading microorganisms and the clearing of apoptotic cells. After internalization, the newly formed phagosome is constantly remodeled by fusion with early endosomes, late endosomes, and lysosomes. These changes ultimately deliver the engulfed material into the terminal degradative compartments known as phagolysosomes. However, defective phagosome maturation can result in inflammatory or autoimmune disease depending on the type of phagosome cargo. Therefore, characterization of the components involved in phagosome formation and maturation is important for a better understanding of macrophage physiological and pathological functions. In this chapter we describe a step-by-step protocol for the isolation of large-scale latex/polystyrene bead phagosome preparations with high degrees of purity for Western blotting analysis of phagosome maturation.
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Anetta
A
Härtlova
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Julien
J
Peltier
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK.
Orsolya
O
Bilkei-Gorzo
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, UK. m.trost@dundee.ac.uk.
Matthias
M
Trost
D000818:Animals; D015153:Blotting, Western / Q000379:methods*; D002458:Cell Fractionation; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D051379:Mice; D008863:Microspheres; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism*; D000067996:RAW 264.7 Cells
Organelle purification; Phagocytosis; Phagosomes; Western blotting