chloroplast import

Dong Sun dcao at blue.weeg.uiowa.edu
Thu Sep 13 11:50:30 EST 2001


Dear All:

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
		    Systems.

	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
import Buffer.

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.

Sincerely







~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Dongsun Cao
Dept. of Biology
The University of Iowa
Iowa City, IA 52242
Tel. 319-335-2583
Fax. 319-335-3620

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