Method Article
Cell-specific mitochondrial affinity purification (CS-MAP) protocol
https://doi.org/10.1038/protex.2017.152
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This step-by step protocol describes the Cell-Specific Mitochondrial Affinity Purification (CS-MAP) Method as described in the Nature Cell Biology paper "Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism"
-NGM plates seeded with E. coli OP50 bacteria.
-M9 Buffer [3 g KH2PO4, 6 g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Sterilize by autoclaving.].
-MIB (Mitochondria Isolation Buffer 50 mM KCl, 110 mM mannitol, 70 mM sucrose, 0.1 mM EDTA (pH 8.0), 5 mM Tris-HCl (pH 7.4), supplemented with protease inhibitors (ref S8830, Sigma)).
-PEB (1 x PBS, 2 mM EDTA, 1% BSA, protease inhibitors).
-anti-HA (Influenza Hemagglutinin) magnetic beads (ref 88836, Pierce).
-A 7 ml dounce homogenizer.
-A homemade setup consisting of a column and a magnet (60 g neodymium).
1- Grow approximately 10,000 worms on 2 x 150 mm NGM plates seeded with E. coli OP50 bacteria.
2- Harvest worms and wash them 3 times with 15 ml of M9 buffer.
3- Wash worms once with 15 ml of ddH2O.
4- Resuspend worms in 3 ml of ice cold MIB and transfer them into a 7 ml dounce homogenizer on ice.
5- Perform 21 gentle strokes.
6- Centrifuge at 200 g for 5 min and keep supernatant.
7- Centrifuge at 800 g for 10 min and keep supernatant.
8- Centrifuge at high-speed 12,000 g for 10 min to pellet the total mitochondria.
9- MIB is removed and replaced by 500 µl of ice cold PEB.
10- Add 100 µg of anti-HA magnetic beads.
11- Incubate samples at 4 °C for 1 h while rotating at 10 rpm.
12- Transfer the samples on a homemade setup consisting of a column and a magnet.
13- Ensure that beads were attracted to the side of the tip and held by the magnet and then wash twice with 25 ml of ice cold PEB.
14- After washings, resuspend the beads in 20 µl of MIB.
Approximately 2 hours
Do not let the bead dry out.
Observation under a fluorescent microscope is used to confirm the presence of fluorescent mitochondria attached to beads.
The authors declare no conflict of interest
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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