I have tried isolating mitochondria from fibroblasts many times using different protocols like C Frezaa,2007;E Gottlieb,2003;Wieckowski,2009;B Antonsson,2000 etc(which slightiy differs from one another in buffer composition and centrifugation speed),but could not get it,even of low purity(tried western blot with TOMM22 and prohibitin as mitochondrial marker).I have used cells harvested from 20-22*100mm plates,either 21 G syringe(passed cell suspension 5-6 times) or Glass-Teflon Potter Elvehjem homogenizer @1000rpm for 5min or combination of both for cell disruption and 2%CHAPS in wash buffer for protein isolation from mitochondrial pellets.Buffers and speed combinations used were-(1a)225mM mannitol,75mM sucrose,0.1mM EDTA,30mM Tris-HCl pH7.4 (1b)resuspesion buffer-250mM mannitol,0.5mM EGTA,5mM HEPES pH7.4(1c)30%percoll(v/v),225mM mannitol,0.1mM EGTA,25mM HEPES pH7.4;7000g,10min;10000g,10min;95000g,30min ;(2)200mM mannitol,70mM sucrose,1mM EGTA,10mM HEPES-NaOH pH7.4 :800g,10 min;10000g,10 min(3)Same buffer with 2000g,3 min;13000g,10min followed by density gradient (1.4-1.6M sucrose) centrifugation @27000rpm for 2 hr in swinging bucket rotor with 5ml tube(4a)Hypo buffer-10mM NaCl,1.5mM MgCl2,10mM Tris-HCl pH7.5 (4b)210mM mannitol,70mM sucrose,1mM EDTA,5mM Tris-HCl pH7.5;1300g,5min;13000g,15 min.
I donot necessarily need very pure mitochondria,but enriched one so that i can have all mitochondrial proteins in my lysed sample.Is there any protocol to isolate mitochondrial proteins from cultured cells without isolating mitochondria?
Have you lyzed the mitochondrial fraction by mild detergent? it should destroy mitochondria and release proteins into solution.
@Tatyana :yes, I lysed mitochondrial fraction(expected) with buffer containing 2%CHAPS or 2%Triton -x -100 followed by sonication at low amplitute (10) for 8-10 seconds.But the problem is in isolation of mitochondria( mainly due to improper homogenization/disruption of cell) not the lysis,i guess.
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