Dear yeasties, I am doing tetrad analysis and can only score one
of the genes by western blotting. This is a membrane bound protein
that is epitobe-tagged with HA. To save time I would like to
grow my tetrads (48 colonies/plate) on Nitrocellulose overlaid
on a YEPD plate (they are growing as I type) and then somehow lyse
the colonies right on the Nitrocellulose. After the protein is
bound to the filter, wash away most of the cell debrie and then probe
the original nitrocellulose disk with anti-HA to score all 12
tetrads. This integral membrane protein (Stv1p) will NOT be secreated
in vivo so I suspect that I will have to some how lyse the cells on the filter.
Has anyone done this? Any ideas or hints on how to lyse whole yeast
colonies on nitrocellulose disks with out smearing smudging etc etc?
Thanks in advance for all advice! Morrie
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Morris F. Manolson Tel: 416-813-6662 (office)
Division of Cell Biology 416-813-5729 (lab)
Hospital for Sick Children 416-813-5028 (FAX)
88 Elm St., McMaster building email: Morrie at resunix.ri.sickkids.on.ca
Toronto, Ontario, Canada
M5G 1X8
