Thus, to ensure a clean muscle sample, it is recommended that
biopsy material is freeze-dried and subsequently dissected free
of visible blood, fat, and connective tissue under the
microscope.- Make up a fresh batch of homogenization buffer and keep it on
ice or at 4C. - Keep the wet weight or freeze-dried tissue on dry ice ( 80 C)
in 2 ml Eppendorf tubes until just before the homogenization
buffer is added. - Open and place the tubes in the TissueLyser plastic racks.
These should be stored at 20 C, so they are cold at all times. - Working quickly, add a 5 mm steel ball to each tube (be careful
that pieces of tissue will not “jump” out of the tube when the
ball hits the bottom). - Add homogenization buffer to all samples in a ratio 20μlto
1 mg of wet weight muscle or 80μl to 1 mg of freeze-dried
muscle (seeNote 4). - Close all tubes and place the rack in the TissueLyser.
- Tissue-lyse two times 1 min at 30 Hz. If there are still large
chunks of muscle in some tubes, tissue-lyse again. - Place the tubes in another rack, and let them rotate end over
end for 1 h at 4C. - Spin down the pellet at 16,000gfor 20 min and 4C.
- Transfer the supernatant (lysate) to a new set of tubes on ice.
- Take out a small portion (10–15μl) for determination of
protein concentration, and store the rest at 80 C. - Use a standard protocol for determination of protein concen-
tration with a bicinchoninic acid (BCA) kit. Dilute the samples
five times in water for the assay.
3.2 Immuno-
precipitation
- Make up a fresh batch of IP buffer and keep it on ice or at 4C.
- Choose the appropriate Protein A or G agarose (agarose beads)
according to the antibody to be used in the immunoprecipita-
tion (IP) (seeNote 2). - Wash the agarose beads thrice in IP buffer, and make up a
50:50 slurry suspension (seeNote 5). - To get the most correct representation of the input to the IP,
make up aPre-IP(seeNote 6). Set up an IP with an irrelevant
antibody that does NOT recognize the protein of interest or
any of its predicted complex partners. This IP is run in parallel
with the “real” IP(s). - Add 20μl of agarose beads (10μl packed beads) to each IP (use
0.2 or 0.5 ml tubes). Keep these tubes on ice.
Analysis of Heterotrimeric Complex Stoichiometry 207