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RiboTAg
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RiboTag Mouse
Fluorescence-activated cell sorting
FACS
~Specialized type of flow cytometry
~Sorts a heterogeneous mixture of
biological cells into two or more
containers, one cell at a time
~Though efficient there is a new alternative....
MACS
How to Sort using RiboTag
Magnetic-activated cell sorting
Wash in Buffer
Immunoprecipitate with Antibody of interest proteins
Magnetic Beads immobilize complexes
Wash unbound complexes
Extract RNAs
End product contains only Ribosome and RNA
RiboTag+Cre=Expression
Isolate tissue (dissect)
Separate cells
Mix cells with HA antibody
Use MACS
RiboTag
RiboTag is a flexible, more cell safe and less
wasteful way to sort cells
What is RiboTag
RiboTag has a modified Ribosomal Protein L22
The Mouse has a hidden triple HA-Tag on its exon 4
RiboTag allows the isolation of Ribosome-associated mRNAs from specific cell types in complex tissues, including brain. (McKnight)
RiboTag is a way to sort cells
Allows less Cell death
Wastes less cells
Flexible
Sources
McKnightLab(http://depts.washington.edu/mcklab/RiboTag.html)
Sanz & Co (RiboTag Analysis of Actively Translated mRNA in Steroli and Leydig Cells in Vivo
Wikipedia
http://www.millipore.com/catalogue/item/17-701#