dcao at blue.weeg.uiowa.edu
Thu Sep 13 11:50:30 EST 2001
I am performing chloroplast import assays. But I did not see my proteins
get imported including positive control (RBCS). The following are the
methods I am using:
Invitro/invitro translation: Promega TnT Coupled Wheat Germ Extract
Chloroplast isolation: 8-10 days pea leaves (Progress 9), only
young leaves are used. They look curley. The intact chloroplasts are
isolated using a Percoll step Gradient(40% Percoll and 85% Percoll, Napier
and Barnes, Methods in Molecular Biology, 49:355).
Import assay: I am using the method described by Perry et al(1991)
50 ug chloroplasts, 4 ul tranlational products, 3ul 100mM MgATP in 150 ul
After import, use a Percoll cushion to recover th chloroplast described by
Perry et al., (1991). Then I run SDS_Page(8% acrilamide) and autoradiograph.
I appreciate it if anyone could give some tips or advices on this
experiment. Thank you in advance.
Dept. of Biology
The University of Iowa
Iowa City, IA 52242
More information about the Arab-gen