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1) which kit works better in isolated primary mouse cardiomyocytes? they will be in suspension from the isolation. 2) In the Q&A, I noticed that supernatant from SPIN2 is considered cytosolic fraction, is this true for both kits? 3) In the ab110170 protocol, it is mentioned that freezing and thawing is recommended. I would like to know whether this is also recommended for primary mouse cardiomyocytes and what temperature was reached for the freezing and thawing (-20C or -80C)? 4) If freezing and thawing is compatible, how long can I store my samples in -20C or -80C from multiple isolations so I can collect all the fractions at once?
Asked on Sep 18 2013
You will want to use either ab110170 or ab110171 (which includes the homogenizer), as these are optimized for isolating fractions from cultured cells versus tissue samples. The supernatant from SPIN2 is the cytosolic fraction, though it might be contaminated with nuclear protein depending on what percentage of nuclei were lysed during the homogenization. The aim is to lyse the plasma membrane by weakening them with the freeze/thaw treatment followed by homogenization, but without damaging the nuclear membrane. The pellet after SPIN1 is ideally intact nuclei.
For the freeze/thaw, we recommend pelleting the cells, aspirating most of the medium, then dipping the vial into liquid nitrogen for one minute and then 37C for one minute or until thawed. The pellet can be stored at this point at -20C or -80C, whichever is more convenient. When you are ready to continue the isolation protocol, add reagent A as instructed in step 3 of the protocol (ab110170). If you do not have liquid nitrogen, store the pellet at -20C or preferable at -80C. When you are ready to isolate mitochondria from all of your samples, thaw the pellets at 37C for one minute to thaw and continue by adding reagent A, step 3.
We do not have data for stability of samples stored at -20 or -80C. Stability of proteins or other biomolecules will depend on what you are investigating, but for the purposes of isolating fractions, we expect this kit should effectively isolate mitochondria from samples stored for several months, if not longer.
Tom RuyleAbcam Scientific Support
Answered on Sep 18 2013