Glass bead vortexing

Cells are broken by adding 4 mm diameter acid-washed glass beads (Thomas Scientific) equal to one-half of the total volume of resuspended cells and rigorously vortexed for 30 s, followed by 1 min on ice, conducted 5 times. Less than 5 cycles were not sufficient for complete cell lysis, whereas more than 5 cycles of vortexing resulted in a significant loss of co-purifying components. 

Add 1.5 ml of chilled glass beads (0.45-0.55 mm) and vortex vigorously in a bead beater by two pulses of 90 s at maximum level to achieve complete lysis. 

Vortex briefly, incubate on ice for 5 min, and centrifuge at max speed in a microcentrifuge for 5 s (to spin the glass beads down). 

Sample preparation 5 Tablets + 2 glass beads + 25 mL 50 g.g/mL dibutyl phthalate in MeOH, vortex 15 min or until tablets have completely disintegrated, sonicate 5 min, filter (2 pm), inject 25 pL aliquot. 

Enzymatic Assay Cells were harvested by centrifugation at 10,000xg for 10 min. The pellet was washed and suspended in buffer (100 mM phosphate buffer, 1 mM EDTA, 5 mM 3-mercaptoethanol [pH 7.0]). The suspended cells were mixed with glass beads (Sigma), vortexed at maximum rate in bursts of 30 to 120 s, and then cooled on ice. The cell debris... 

Resuspend cell pellets in 100 pL of STET buffer, add a 150 pL vol of acid-washed glass beads, and disrupt the cells by vigorous vortexing for 5 min... 

Add 50-100 pi of acid-washed glass beads and vortex for 5 min. Let it stand to allow the beads to setde. Transfer supernatant to a fresh 1.5-ml microcentrifuge tube. 

To get rid of the unmodifled peptides adsorbed to the glass surface, wash the pelleted SPH beads with 400 pL of reaction buffer. Repeat this step three more times. Subsequent washes should be performed each with 400 pL of 1 M NaCl (four times, 4x), distilled water (4x), 80% acetonitrile (4x), and a final wash by distilled water (4x). Each washing step involves a mild vortexing (30 s) followed by a brief centrifugation to settle the beads and changing the wash solution. 

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