Sediment slurry (250 μl) was diluted with 150 μl of 2.5% NaCl solution, and 50 μl of detergent mix [100 mM EDTA, 100 mM sodium pyrophosphate, 1% (v/v) Tween 80] and 50 μl methanol were added. Next, the sample was vigorously shaken for 60 min at 500 r.p.m. using a Shake Master (Bio Medical Science, Tokyo, Japan). After shaking, the sediment slurry was sonicated at 20 W for 1 min using a Model UH-50 Ultrasonic Homogenizer (SMT, Tokyo, Japan), and then carefully layered onto a high-density cushion solution. Either 50% (w/v) Nycodenz (Kallmeyer et al., 2008 ) or various combinations of 50–80% (w/v) Nycodenz and 40–80% (w/v) sodium polytungstate were used to prepare the high-density solutions. Samples were centrifuged at either 4500× g or 15 000× g for 15–300 min, after which the supernatant, including the high-density layer(s), was carefully removed and transferred to a separate vial. Next, 900 μl of 2.5% NaCl solution was added to the remaining high-density solution and sediment pellet, which was resuspended and centrifuged again at 5000× g for 15 min. The resulting supernatant was also transferred to a separate vial. The remaining pellet was resuspended in 100 μl of 1% hydrofluoric acid and allowed to stand for 20 min. The reaction was stopped by adding 100 μl of 1.5 M Tris-base, and the sample was shaken again for 10 min after addition of 150 μl of 2.5% NaCl solution and 50 μl each of detergent mix and methanol. The vial was then sonicated in a water bath for 30 s, and layering onto the high-density solution and subsequent centrifugation steps were repeated as described above.
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