Disruption of Nematodes
Protein Extraction of Nematode Worms
Christopher E Hopkins, PhD
Knudra Transgenics (www.knudra.com)
The following lysis procedure provides >90 efficiency in releasing a cytoplasmic protein (for example, UNC-18) into the soluble fraction of a MiniBeadbeater lysate of nematodes:
Sucrose-gradient cleaned worms are centrifugally pelleted, then resuspend in ice-cold Lysis Buffer (M9 salts, 1% trition, 1x protease inhibitors (Roche)) at 2x the pellet volume (50% worms). A MiniBeadbeater microvial is filled to the 0.7 ml mark with 1 mm zirconia beads and the 50% worms are added to fill the vial to top ( approx. 1400 ul of 50% worms used). The vial is capped, added to MiniBeadbeater and beadbeat at the maximum speed setting for 20 seconds. The vial is removed and immersed into moving ice/ water mix ( stir bar at approx. 200 rpm) for one minute. The beadbeating and ice water cooling is repeated 6 more times (2 min total processing time). Worm lysate supernatant is recovered by centrifugation at approx. 10K g for 10-20 min.
Temperature measurements revealed a 20 second pulse caused a 12-14 degree increase in temperature. A one minute ice water treatment returned the vial to nearly zero degrees. Other experiments demonstrated the time for nematode death (LD50) occured within 10 seconds of beadbeating.
One millimeter beads made of glass were 3x slower than the same size zirconia beads in achieving the same lysis efficiency. When 0.1mm glass beads were tried, most of the worms were not killed in a reasonable time (less than 1 min).