Mitochondrial Swelling Protocol

Paul S. Brookes. brookes at uab.edu
Tue Nov 23 13:24:14 EST 1999


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The protocol published by Packer and Murphy a while back (96) works well in 
our hands with liver or heart mitochondria.  You might try using less HEPES 
in the buffer, depending on the pH effects of what you're adding to induce 
pore opening.  Also be very careful to reduce te EGTA in your mito' prep' 
so you don't bind any Ca2+ you add.  Make sure you check that what you're 
looking at is cyclosporin A sensitive.  Be mindful of adding things that 
change the spectra too, as 540nm is awfully close to the redox spectra 
region of the cytochromes.

After the swelling, spin the mito's in a microfuge FOR AT LEAST 15 MINUTES 
!!!!!!!
I've seen papers where they spin for 3 min's and then look for cyt-c by its 
spectra - we can show this, but if you spin for 15 there's nothing there, 
so basically they're looking at cyt-c in non-pelleted mitochondria.  Of 
course, swelling will change the buoyancy of the mito's, so swollen ones 
don't pellet so well.   If you spin for 15 min's and use a standard WB 
protocol and a 15% gel its easy to detect cyt-c release.

Good luck
Paul


_________________________________________
Dr. Paul S. Brookes.            (brookes at uab.edu)
UAB Department of Pathology,   G004 Volker Hall
1670 University Blvd., Birmingham AL 35294 USA
Tel (001) 205 934 1915     Fax (001) 205 934 1775
http://peir.path.uab.edu/brookes

The quality of e-mails can go down as well as up

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<html><div>The protocol published by Packer and Murphy a while back (96)
works well in our hands with liver or heart mitochondria.&nbsp; You might
try using less HEPES in the buffer, depending on the pH effects of what
you're adding to induce pore opening.&nbsp; Also be very careful to
reduce te EGTA in your mito' prep' so you don't bind any Ca2+ you
add.&nbsp; Make sure you check that what you're looking at is cyclosporin
A sensitive.&nbsp; Be mindful of adding things that change the spectra
too, as 540nm is awfully close to the redox spectra region of the
cytochromes.</div>
<br>
<div>After the swelling, spin the mito's in a microfuge FOR AT LEAST 15
MINUTES !!!!!!!</div>
<div>I've seen papers where they spin for 3 min's and then look for cyt-c
by its spectra - we can show this, but if you spin for 15 there's nothing
there, so basically they're looking at cyt-c in non-pelleted
mitochondria.&nbsp; Of course, swelling will change the buoyancy of the
mito's, so swollen ones don't pellet so well.&nbsp;&nbsp; If you spin for
15 min's and use a standard WB protocol and a 15% gel its easy to detect
cyt-c release.</div>
<br>
<div>Good luck</div>
<div>Paul</div>
<br>
<br>

<font color="#000080">_________________________________________<br>
</font><font color="#FF0000"><b>Dr. Paul S.
Brookes.</b>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
(brookes at uab.edu)<br>
</font><font color="#000080">UAB Department of Pathology,&nbsp;&nbsp;
G004 Volker Hall<br>
1670 University Blvd., Birmingham AL 35294 USA<br>
Tel (001) 205 934 1915&nbsp;&nbsp;&nbsp;&nbsp; Fax (001) 205 934
1775<br>
<a href="http://peir.path.uab.edu/brookes" eudora="autourl">http://peir.path.uab.edu/brookes</a><br>
<br>
<b>The quality of e-mails can go down as well as up<br>
</font></b></html>

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