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The acidic SL forms get eluted first while the lactonic SLs, especially the acetylated ones, show longer retention times because of higher hydrophobicity [ 24].
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After removing the solvent under reduced pressure, 1390.5 g ethyl acetate fraction was subjected to silica gel column chromatography (CC) and was eluted to get fractions 1 11 using a step-gradient solvent system of ethyl acetate (EA):petroleum ether (PE) (0 100 → 100 0).
Ethanol precipitation is not required in order to get rid of the eluting salt.
Subfraction VII-3 was separated by RP-MPLC, eluted with MeOH/H2O (20:100–55:100) to get subfractions VII-3-1 and VII-3-2.
DNA was eluted with an elution buffer.
Fr.2 (60 g) was then chromatographed on a silica gel column eluted with petroleum ether/acetone (25:1), to get 2 (0.6 g), 3 (45 mg) and Fr.2-3.
The RNA was eluted in 50 µl elution buffer.
Protein was eluted with 1× denaturing elution buffer (Millipore).
This implies that, assuming the distributions displayed in Figure 2, the peptides eluting in the middle of the run get a lower probability to be selected for fragmentation, and thus identified.
Fr3-3 was chromatographed on a silica gel column eluted with petroleum ether/acetone (15:1), and semipreparative HPLC to get 8 (19 mg) and 9 (16 mg).
The RNA was eluted in 60 µl of elution buffer.
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