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. 2001 Sep 18;98(20):11205–11210. doi: 10.1073/pnas.211316798

Figure 3.

Figure 3

HPLC purification of allurin. (A) HPLC purification of FPLC fraction 3. Elution profile of fraction 3 applied to a C-3 RP-HPLC column and eluted with acetonitrile. Allurin elutes as a sharp peak at 60% acetonitrile. SDS/PAGE analysis of HPLC-purified allurin is shown in the Inset (arrowhead). Protein standards at 200, 116, 97, 66, 55, 36, 31, 21, 14, and 6 kDa are shown in the lane to the left. (B) MALDI-TOF spectrum of HPLC peak. MALDI-TOF MS of HPLC-purified allurin. Single-, double-, and triple-charged molecular ions are seen at 21,053, 10,534, and 7,023 Da per electron unit, respectively. Cytochrome C, used as an internal standard, exhibits single- and double-charged molecular ions at 12,361 and 6,183 Da per electron unit, respectively. Because of matrix adduct-induced broadening of the single-charged molecular ion peak, the most accurate estimate of molecular mass is considered to be 21,068 Da, which is derived from the double-charged molecular ion peak. (C) Chemotaxis activity during purification. Dose-response curves for sperm chemoattractant activity in whole egg jelly (◊), 12HEW (▵), FPLC fraction 3 (○), and HPLC-purified allurin (□). Data points represent means of 3 replicate experiments. All standard errors are less than 10% of the mean.