westerns on yeast colonies

Morris F. Manolson mm6y+ at andrew.cmu.edu
Thu Apr 22 12:58:20 EST 1993


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



******************************************************************************
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



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