Abstract
Previously, it has been shown that phospholipids, cholesterol, and glycolipids could be quantitated using the same high performance thin-layer chromatography (HPTLC) method. Here we examined that method in terms of linearity of standards in the nanogram range, recovery of nonacidic and acidic lipids after Sephadex column chromatography, and quantitation of lipids in mouse synaptic plasma membranes (SPM) where lipid content is low. Nonacidic and acidic fractions were separated by Sephadex column chromatography, applied to plates using contact spotting, chromatographed, visualized with cupric acetate, and quantitated using in situ densitometry. Recovery of nonacidic and acidic fractions off the columns was determined with radiolabeled phospholipids. Standards for each lipid class were linear in the nanogram range. Quantitation of SPM lipid classes could be made with as little as 1.5 micrograms of total lipid. Recovery of the nonacidic fraction after Sephadex column chromatography was approximately 100% whereas the acidic fraction was approximately 91%. Phospholipids, cholesterol, and glycolipids could be determined in nanogram amounts using the same method. This method is an efficient method for examining different lipid classes and in samples where lipid content is low.
Highlights
The 0.95 M sucrose-water interface was removed and sedimented at 40,000 g for 20 min, and the final synaptic plasma membranes (SPM) pellet was resuspended in 1 ml of 50 mM Tris, pH 7.3
SPM lipids were extracted in 4 volumes of chloroform-methanol 2:l (v/v) and centrifuged at 750 g for 10 min [13]
SPM lipid content is described in Table 2 and Table 3
Summary
Phospholipid, cholesterol, and glycolipid standards were obtained from Supelco (Bellefonte, PA) and Sigma Chemical Company The 0.95 M sucrose-water interface was removed and sedimented at 40,000 g for 20 min, and the final SPM pellet was resuspended in 1 ml of 50 mM Tris, pH 7.3. SPM lipids were extracted in 4 volumes of chloroform-methanol 2:l (v/v) and centrifuged at 750 g for 10 min [13]. The acidic fraction was brought up in chloroform-methanol 2:l and 0.6 ml of water and centrifuged This procedure is necessary to remove the sodium acetate. The upper layer was removed, discarded, and an equal amount of chloroform- methanol-water 3:48:47 was added, and the mixture was centrifuged. Separate HPTLC plates for the acidic and nonacidic lipids were developed using chloroform-methanol-acetic acid-formic acid-water 35:15:6:2:1 (v/v).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
