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?

More Janette Tong's questions See All
Similar questions and discussions