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.
harkness at bio.bu.edu