Phytoceramide in Vertebrate Tissues: One Step Chromatography Separation for Molecular Characterization of Ceramide Species
Figure 1
Thin-layer chromatography of silicic acid purified Cer fractions from astrocytes primary culture and from mouse brain lipid extract.
A. The Cer fractions (using previous method) from astrocytes were applied to the HPTLC plate and developed using chloroform:methanol:acetic acid (95:5:0.5, v/v/v). Ceramide bands were visualized after iodine absorption. Lanes 1–3: Std. NFACer; Lane 4–7: Ceramide fractions isolated from astrocytes; Lane 8: Cholesterol; Lane 9: Std. HFACer. Note: The appearance of contaminants (cholesterol) in all preparations. B. Pooled Cer fractions from astrocytes (1A) were applied on a silicic acid column and eluted with solvent choloroform;acetone:acetic acid (24:10.01, v/v/v, solvent 1). After eluting the column with 10 ml of the solvent 1, the column was eluted using choloroform;acetone:acetic acid (18∶2∶0.01, v/v/v, solvent 2). Approximately 1 ml fractions were collected and 10 µl was applied on the TLC plate and developed using chloroform:methanol:acetic acid (95∶5:0.5, v/v/v). The bands were visualized using char spray [6]. Lanes 1–2: Fractions collected using solvent 1 (approximately 5 ml each); Lanes 3–5, 7–12 fractions (1 ml each) collected using solvent 2; Lane 6: Std. NFACer. C. The purified Cer fractions (F2) from mouse brain were dissolved in chloroform:acetone 19:1. Fifteenµl was applied on each lane and bands were separated using chloroform:methanol:acetic acid (95:5:0.5, v/v/v) and visualized after iodine absorption as described in the text. Lanes 1–6: Ceramide fractions from brain; Lanes 7–9: Std. NFACer.