I am about to embark on a subcellular localization study of a specific
protein in yeast. I know that this topic has been flogged a bit in the
past on this news group but after going through the biosci archives, I
still have a few questions.
1) What is the final consensus regarding GFP-fusions in cerevisiae? I know
that some mutants of GFP are available which improve the signal to noise
ratio, but of these, which are now considered to be the best? Does anyone
out there have a good yeast expression vector which will place GFP at the
N-terminus (or C-terminus) of a protein of interest? Also, is anyone
willing to share a similar expression vector with a triple HA-tag?
2) Can anyone direct me to a good protocol for subcellular fractionation
I will be happy to summarize the information I receive on this topic and
post it to the entire group.
Thanks in advance.