Optimal OD's for westerns or beta-gals?

Daniel Harkness harkness at bu.edu
Thu Aug 14 14:24:13 EST 1997


John Stebbins (stebbin1 at pilot.msu.edu) wrote:
: I just purchased the Clontech lexA mAB and attempted to do a western with
: it which failed.  One reason given for the failure was that the yeast were
: collected at OD600=1.0 instead of 0.5.  

Similar questions have been raised before and I always get a gut feeling
about what might be going in these cases: not enough cells, not enough 
starting material.  When I perform a western looking for my protein (ADH
promoter/CEN) I typically use 50mls of cells (OD=~1.0).  I spin, transfer to
an eppindorf, add .3gms glass beads, add ~500ul of 1X SDS-sample buffer and
vortex for 5 minutes.  I load 40-50ul onto my gel.

Needless to say, when I was using 5ml cultures my results were "negative".
I hope this is at all helpful.

-Dan Harkness
harkness at bio.bu.edu




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