No protein is visible from any of my sucrose fractions following this method:
I used 3-5 mg of mitochondria for osmotic shock and sonication, followed by a 14,000 rpm, 10 min centrifugation to clear out intact mitochondria. The supernatant was centrifuged at 100,000g for 60 mins to pellet membranes. Membrane pellet was resuspend in 400 ul loading buffer (5 mM K+/Hepes pH 7.4, protease inhibitors, 10 mM KCl) and spin at 14,000 rpm, 10 mins. The supernatant was loaded on to sucrose gradient with 1 ml 1.6M sucrose, 5 ml 1.35M sucrose, 2.5 ml 1.1M sucrose and 1.5 ml 0.85M sucrose, followed by centrifugation at 100,000g for 16 hrs at 4 deg. 750 ul fractions were collected post-spin and incubated with final 25% of TCA for 1 h on ice. Precipitated proteins were collected by centrifugation at 14,000 g for 30 minutes at 4 deg and washed with ice-cold acetone, air dry and loaded onto 12% tris- glycine (gel ran all the way). Immunoblotting of PVDF (transfer is successful with markers and positive control) was carried out with abundant outer membrane (porin) and inner membrane (Tim23) proteins. The positive control is 25 ug purified mitochondria. I could see both porin and Tim23 in the control lane, but nothing (not even background) in all of the sucrose fractions.
Could anybody help me with this problem?
Hi Janette, I didn't catch the point, what do you want to obtain? Inner and outer membrane proteins? or just integral membrane proteins w/o distinction of inner and outer? Digitonin treatment or sodium carbonate should be easier method.
Janette, try the method well described here:
Hallermayer G, Neupert W. Lipid composition of mitochondrial outer and inner membranes of Neurospora crassa.
Hoppe Seylers Z Physiol Chem. 1974; 355(3): 279-88.
Janette, conditions for osmotic shock (followed by a bit of homogenization) need to optimized for different tissues and cell types. You could check Douce et al BBA 292 (1973) 105 for details.
I have done my phD thesis on that...
This is a long work at the bench... for obtaining outer membrane, the yield is low but the literature is clearly available....
For the inner membrane you wil get inside-out vesicle or inside out vesicles depending of the procedure used, i.e. sonication or French press in medium of different ionic strength medium
it also depend if you started from plant tissues or animal tissus.
I agree with Carmen Manella.. R. Douce al. (1997) and other from the same lab. Could be useful.. If you do not consider that this is related to before -JC....
Most of best work with isolated membrane comes from the ancient time when research was a work and time was given...
I can send you the publications if needed... Patrice
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