TABLE 2.

Extraction of polyP using silicate glass^a

Fraction	Recovery (%)
	PolyP (chain length)			ATP
	60–300	100–500	750–800
Unbound	5	2	0	93
Wash	3	3	2	7
Eluted	92	95	98	0

^aE. coli CF5802 (0.5 ml of a culture with an OD₆₀₀ of 1.7) was lysed in 0.3 ml of 4 M GITC lysis buffer and kept at 95°C for 2 min and then sonicated for 5 min; 5 μl of [³²P]polyP (7 nmol/mg of protein, final concentration) or [γ-³²P]ATP (258 nmol/mg of protein, final concentration), 30 μl of 10% SDS, 0.3 ml of 95% ethanol, and 5 μl of Glassmilk were sequentially added, the glass was pelleted, and the supernatant was collected (unbound). The glass pellets were resuspended in New Wash buffer by sonication and pelleted, the supernatant was collected, the wash was repeated, and the supernatants were pooled (wash). The washed pellet was then resuspended in 50 μl of 50 mM Tris-HCl (pH 7.4)–10 mM MgCl₂–20 μg each of DNase and RNase per ml and incubated at 37°C for 10 min. The pellet was washed first with 150 μl of 4 M GITC lysis buffer and 150 μl of 95% ethanol and then twice in New Wash buffer. PolyP was eluted from the pellet with 50 μl of 50 mM Tris-HCl (pH 8.0) at 95°C for 2 min; recovery of polyP was complete with two additional elutions (eluted). Values are averages of duplicates and were within 2% of each other.