Separation of (Phospho)Lipids by Thin-Layer Chromatography

Fuchs, Beate; Popkova, Yulia; Süß, Rosmarie; Schiller, Jürgen

doi:10.1016/b978-0-12-417223-4.00014-5

Cited by 5 publications

(2 citation statements)

References 97 publications

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“…TLC analysis of lipids was performed essentially as described in [81]. Lipid extracts were dissolved in chloroform and spotted on activated silica gel plates.…”

Section: Lipid Analysis By Tlcmentioning

confidence: 99%

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

Yang

et al. 2020

BMC Biol

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Background: When stressed, eukaryotic cells produce triacylglycerol (TAG) to store nutrients and mobilize autophagy to combat internal damage. We and others previously reported that in yeast, elimination of TAG synthesizing enzymes inhibits autophagy under nitrogen starvation, yet the underlying mechanism has remained elusive. Results: Here, we show that disruption of TAG synthesis led to diacylglycerol (DAG) accumulation and its relocation from the vacuolar membrane to the endoplasmic reticulum (ER). We further show that, beyond autophagy, ERaccumulated DAG caused severe defects in the endomembrane system, including disturbing the balance of ER-Golgi protein trafficking, manifesting in bulging of ER and loss of the Golgi apparatus. Genetic or chemical manipulations that increase consumption or decrease supply of DAG reversed these defects. In contrast, increased amounts of precursors of glycerolipid synthesis, including phosphatidic acid and free fatty acids, did not replicate the effects of excess DAG. We also provide evidence that the observed endomembrane defects do not rely on Golgi-produced DAG, Pkc1 signaling, or the unfolded protein response. Conclusions: This work identifies DAG as the critical lipid molecule responsible for autophagy inhibition under condition of defective TAG synthesis and demonstrates the disruption of ER and Golgi function by excess DAG as the potential cause of the autophagy defect.

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“…TLC analysis of lipids was performed essentially as described in [81]. Lipid extracts were dissolved in chloroform and spotted on activated silica gel plates.…”

Section: Lipid Analysis By Tlcmentioning

confidence: 99%

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

Yang

et al. 2020

BMC Biol

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“…propanol or hexane have also been utilized(Fuchs et al, 2015;Kohlwein, 2017a and2017b). In this protocol, we use CHCl3/CH3OH (2:1 v/v) as the solvent for lipid extraction and break yeast cells with glass beads in a mechanical bead mill.…”

mentioning

confidence: 99%

Yeast Lipid Extraction and Analysis by HPTLC

Li¹,

Zhang²,

He³

et al. 2021

BIO-PROTOCOL

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The diversity of lipid structures, properties, and combinations in biological tissues makes their extraction and analysis an experimental challenge. Accordingly, even for one of the simplest singlecellular fungi, the budding yeast (Saccharomyces cerevisiae), numerous extraction and analysis protocols have been developed to separate and quantitate the different molecular lipid species. Among them, most are quite sophisticated and tricky to follow. Herein, we describe a yeast total lipids extraction procedure with a relatively good yield, which is appropriate for subsequent thin-layer chromatography (TLC) or liquid chromatography-mass (LC-MS) analysis. We then discuss the most widely used solvent systems to separate yeast phospholipids and neutral lipids by TLC. Finally, we describe an easy and rapid method for silica gel staining by a Coomassie Brilliant Blue-methanol mixture. The stained lipid species can then be quantitated using imaging software such as ImageJ. Overall, the methods described in this protocol are time-saving and novice-friendly.

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Detection and Semi‐Quantification of Lipids on High‐Performance Thin‐Layer Chromatography Plate Using Ceric Ammonium Molybdate Staining

Asressu

Zhang

2022

Euro J Lipid Sci & Tech

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It is desirable to quickly check the composition of lipids in small size samples, but achieving this is challenging using the existing staining methods. Herein, a highly sensitive and semi‐quantitative method is developed for analysis of lipid samples with ceric ammonium molybdate (CAM) staining. The CAM detection method is systematically evaluated with a wide range of lipid classes including phospholipids, sphingolipids, glycerolipids, fatty acids (FA), and sterols, demonstrating high sensitivity, stability, and overall efficiency. Additionally, CAM staining provides a clean yellow background in high performance thin‐layer chromatography (HPTLC) which facilitates quantification of lipids using image processing software. Lipids can be stained with CAM reagent regardless of their head group types, position of the carbon–carbon double bonds, geometric isomerism, and the variation in the length of FA chain, but staining is mostly affected by the degree of unsaturation of the FA backbone. The mechanism of the CAM staining of lipids is proposed on principles of the reduction–oxidation reaction, in which Mo(VI) oxidizes the unsaturated lipids into carbonyl compounds on the HPTLC plate upon heating, while itself being reduced to Mo(IV). This method is applied for the separation, identification, and quantification of lipid extracts from porcine brain. Practical applications: The CAM staining method that is developed in this work shows a high sensitivity for diverse lipid classes following HPTLC separation. HPTLC with CAM staining is a promising method for quick assessment of the identity and quantity of diverse lipid classes in lipid extracts of small size biological samples.

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Separation of (Phospho)Lipids by Thin-Layer Chromatography

Cited by 5 publications

References 97 publications

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

Yeast Lipid Extraction and Analysis by HPTLC

Detection and Semi‐Quantification of Lipids on High‐Performance Thin‐Layer Chromatography Plate Using Ceric Ammonium Molybdate Staining

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