[Arabidopsis] Re: suspension cell culture protein isolation

rodnay rodnay at gmail.com
Fri May 12 04:49:08 EST 2006


Hi,
Tissue samples of 0.2 or 0.5 g from Arabidopsis and other plants were
frozen in liquid N2 and ground to a fine powder. The powdered samples
were mixed with 23 ml of freshly prepared extraction buffer (25 mm
TrisHCl pH 7.5, 10 mm NaCl, 10 mm MgCl2, 5 mm EDTA, 10 mm
-mercaptoethanol, 1 mm PMSF, 0.2 mg ml 1 benzidine and 0.2 mg ml 1
leupeptin). The homogenates were centrifuged (15 000  g ) for 15 min
to remove the debris. TCA was added to the supernatant to a final
concentration of 10% to precipitate the proteins. After 1 h incubation
on ice, the samples were centrifuged and the pellet was washed twice
with ice-cold acetone, dried, and dissolved in 100 l (0.2 g tissue)
or 250 l (0.5 g tissue) of solubilization buffer (11 mg Na2CO3
per ml 100 mm DTT). The samples were sonicated briefly (2  15 sec
at 40 W) on ice to solubilize the pellet.
good luck for your experiment
rod



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