Hello,
I am a graduate student am having problems with a GST tag, it appears
to be binding to purified golgi. The GST tag is on the N terminus of
my protein(myosin) in a yeast expression vector, driven by GAL
promoter. The protein is expressed in high levels, sequence of the
vector is correct and the GST is recognized by an anti-GST antibody.
After trying to purify the expressed protein without success I decided
to use the yeast lysate. The yeast are broken open by vortexing with
glass beads, and whole cells and debris removed by high speed
centrifugation. The lysate is incubated with Golgi, in 150 mM salt,
the Gogli is separated by floating up through a step gradient.
removing the Golgi first, and western analysis shows my tag in the
golgi fraction. it is not contamination.
any ideas would be appreciated.
Kathleen
