discriminating SOD1 and SOD2 activities

cloningman xxxgifalone at tiscali.it
Wed May 31 02:33:06 EST 2006


Hi,
in our laboratory we are trying to discriminate Cu/Zn-SOD and Mn-SOD
activities. We're trying with the Chloroform/ethanol extraction. We follow
this protocol:
organ (rat brain) is homogenized by pottering, ultracentrifuged for 60 mins
at 4°C and then recovered supernatant is splitted in two parts. One is
treated with 0,4 volumes chloroform/ethanol and immediately centrifuged at
13000 rpm (4°C). The other part is not treated and follows the same
centrifugation step. Organic extraction, according to what published in some
works, inactivate only the Mn-SOD, leaving inalterate the Cu/Zn-activity.

Then upper phase (acqueous phase) is recovered and immediately assayed for
SOD enzymatic activity (Sun & Zigman, 1978) and for protein content
(Bradford method).
We get the following issue:
1) in organic-extracted samples we get a 10-fold protein content decrease
(expressed as mg/mL of sample);
2) in organic-extracted samples SOD activity show a 20% decrease (expressed
as mU/mL of sample).

Now, since I get a dramatic fall in protein content, it's impossibile for me
to compare specific activities after and before organic treatment. Is it
possibile to establish Mn-SOD contribution comparing mU/mL activities in non
treated and treated samples?

I hope I've been clear. Thanks in advance to everyone.

-- 
Cloningman

xxxgifalone at tiscali.it (remove xxx)




More information about the Methods mailing list