Hello all,
does anyone know which is a better plasma membrane marker for expression in
N.benthamiana for confocal observation? I am studying a protein kinase,
which is predicted to localize at PM because it harbors palmitoylation site. I
need to co-localize my GFP-protein with RFP-tagged marker.
I have used PIP2A(*AT3G53420)*, which is a aquaporin protein of PM,
according to
Brook K. Nelson†, Xue Cai†,‡ and Andreas Nebenfu¨ hr. A multicolored set of
in vivo organelle markers for co-localization studies in Arabidopsis and
other plants. The Plant Journal (2007) 51, 1126–1136
and
GenBank: X75883.1
AUTHORS Kammerloher,W., Fischer,U., Piechottka,G.P. and Schaffner,A.R.
TITLE Water channels in the plant plasma membrane cloned by
immunoselection from a mammalian expression system
JOURNAL Plant J. 6 (2), 187-199 (1994)
but it is not good in my experiment, as it also resided in the cytosol and
envelop of some nuclei, which is quite wierd to me.
I used double CaMV 35S to drive the expression of both my gene and the
marker genes.
In another assay with a nuclear protein, I used VIP1(a bZIP TF） as the
nuclear marker, again, it is so bad. both cytosol(seems PM too） and nuclei
showed very strong red signal.
reference: Tzfira, T., Vaidya, M. and Citovsky, V. 2001. VIP1, an Arabidopsis
protein that interacts with Agrobacterium VirE2,is involved in VirE2
nuclear import and Agrobacterium infectivity. EMBO J. 20: 3596–3607.
any solution and suggestion? Is it because the expression level of marker
gene too high as a result of using double CaMV35S promoter? It should be
known that most nowsdays binary vector bear double CaMV35S promoter to
drive both target gene and selection marker gene.
thanks in advance,
smith
