Differential centrifugation-based biochemical fractionation of the Drosophila adult CNS.
Drosophila is widely used as a genetic model in questions of development, cellular function and disease. Genetic screens in flies have proven to be incredibly powerful in identifying crucial components for synapse formation and function, particularly in the case of the presynaptic release machinery. Although modern biochemical methods can identify individual proteins and lipids (and their binding partners), they have typically been excluded from use in Drosophila for technical reasons. To bridge this essential gap between genetics and biochemistry, we developed a fractionation method to isolate various parts of the synaptic machinery from Drosophila, thus allowing it to be studied in unprecedented biochemical detail. This is only possible because our protocol has unique advantages in terms of enriching and preserving endogenous protein complexes. The procedure involves decapitation of adult flies, homogenization and differential centrifugation of fly heads, which allow subsequent purification of presynaptic (and to a limited degree postsynaptic) components. It is designed to require only a rudimentary knowledge of biochemical fractionation, and it takes ∼3.5 h. The yield is typically 4 mg of synaptic membrane protein per gram of Drosophila heads.
Institute for Biology - Genetics, Freie Universität Berlin, Berlin, Germany.
Harald
H
Depner
Institute for Biology - Genetics, Freie Universität Berlin, Berlin, Germany.
Janine
J
Lützkendorf
Institute for Biology - Genetics, Freie Universität Berlin, Berlin, Germany.
Husam A
HA
Babkir
1] Institute for Biology - Genetics, Freie Universität Berlin, Berlin, Germany. [2] NeuroCure Cluster of Excellence, Charité, Berlin, Germany.
Stephan J
SJ
Sigrist
Laboratory of Glia Biology, Vlaams Instituut voor Biotechnologie (VIB) Center for the Biology of Disease, Katholieke Universiteit (KU) Leuven, Leuven, Belgium.
Matthew G
MG
Holt
D000818:Animals; D002490:Central Nervous System / Q000737:chemistry*; D002498:Centrifugation / Q000379:methods*; D005591:Chemical Fractionation / Q000379:methods*; D004330:Drosophila / Q000737:chemistry*; D029721:Drosophila Proteins / Q000737:chemistry / Q000302:isolation & purification; D006257:Head; D015203:Reproducibility of Results; D013569:Synapses / Q000737:chemistry*; D009435:Synaptic Transmission; D013574:Synaptosomes / Q000737:chemistry; D057188:Workflow
D029721:Drosophila Proteins