Analysis of protein transport to lysosomes.
Lysosomes are terminal degradative organelles that are found in all higher eukaryotic cells. The biogenesis of lysosomes involves the transport of various acid hydrolases and transmembrane glycoproteins from their site of synthesis in the endoplasmic reticulum through the biosynthetic and endocytic pathways. Protein transport to lysosomes can be studied by a combination of techniques based on the separation of intracellular organelles. Percoll density gradient centrifugation has long been the method of choice for separating lysosomes from other organelles in cell homogenates, and accordingly, this unit describes protocols for obtaining reasonably pure lysosomal fractions from mammalian cells using Percoll density gradient separation.
Schaub
Beat E
BE
University of Zurich, Zurich, Switzerland.
Nair
Prashant
P
University of Zurich, Zurich, Switzerland.
Rohrer
Jack
J
University of Zurich, Zurich, Switzerland.
D000818:Animals; D001671:Biochemistry / Q000379:methods*; D018929:Cell Culture Techniques; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D011205:Povidone / Q000737:chemistry; D021381:Protein Transport / Q000502:physiology; D012822:Silicon Dioxide / Q000737:chemistry; D013347:Subcellular Fractions / Q000378:metabolism
C016039:Percoll; D012822:Silicon Dioxide; D011205:Povidone