Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity

Published: April 30, 2023

Abstract

Source: Guallar-Garrido, S., et alAnalysis of the Lipid Composition of Mycobacteria by Thin Layer Chromatography. J. Vis. Exp. (2021).

This video demonstrates the thin-layer chromatography-based separation of mycolic acids. This technique separates the different mycolic acids—long-chain lipids present in the cell wall of mycobacteria—based on their polarity.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Extraction of the total non-covalent-linked lipids from mycobacteria (Figure 1) Scratch 0.2 g of mycobacteria from a solid media and add to a glass tube with a polytetrafluoroethlene (PTFE) liner screw caps. Add a solution consisting of 5 mL of chloroform and 10 mL of methanol (chloroform:methanol, 1:2). NOTE: Wh…

Representative Results

Figure 1: Scheme of the procedure of extracting lipid content of mycobacteria grown on solid media. Main steps to decipher lipids present on mycobacteria cells. Figure 2: Scheme of the procedure for extracting mycolic acid content of mycobacteria grown on solid media. Main steps to…

Declarações

The authors have nothing to disclose.

Materials

Acetic Acid Merck 100063 CAUTION. Anhydrous for analysis EMSURE® ACS,ISO,Reag. Ph Eur
Acetone Carlo Erba 400971N CAUTION. ACETONE RPE-ACS-ISO FOR ANALYS ml 1000
Anthrone Merck 8014610010 Anthrone for synthesis.
Benzene Carlo Erba 426113 CAUTION. Benzene RPE – For analysis – ACS 2.5 l
Capillary glass tube Merck BR708709 BRAND® disposable BLAUBRAND® micropipettes, intraMark
Chloroform Carlo Erba 412653 CAUTION. Chloroform RS – For HPLC – Isocratic grade – Stabilized with ethanol 2.5 L
Dry block heater J.P. Selecta 7471200
Dicloromethane Carlo Erba 412622 CAUTION. Dichloromethane RS – For HPLC – Isocratic grade – Stabilized with amylene 2.5 L
Diethyl ether Carlo Erba 412672 CAUTION. Diethyl ether RS – For HPLC – Isocratic grade – Not stabilized 2.5 L
Ethyl Acetate Panreac 1313181211 CAUTION. Ethyl acetate (Reag. USP, Ph. Eur.) for analysis, ACS, ISO
Ethyl Alcohol Absolute Carlo Erba 4146072 CAUTION. Ethanol absolute anhydrous RPE – For analysis – ACS – Reag. Ph.Eur. – Reag. USP 1 L
Glass funnel VidraFOC DURA.2133148 1217/1
Glass tube VidraFOC VFOC.45066A-16125 Glass tube with PTFE recovered cap
Methanol Carlo Erba 412722 CAUTION. Methanol RS – For HPLC – GOLD – Ultragradient grade 2.5 L
Molybdatophosphoric acid hydrate Merck 51429-74-4 CAUTION
Molybdenum Blue Spray Reagent, 1.3% Sigma M1942-100ML CAUTION
n-hexane Carlo Erba 446903 CAUTION. n-Hexane 99% RS – ATRASOL – For traces analysis 2.5 L
n-nitroso-n-methylurea Sigma N4766 CAUTION
Orbital shaking platform DDBiolab 995018 NB-205L benchtop shaking incubator
Petroleum ether (60-80ºC) Carlo Erba 427003 CAUTION. Petroleum ether 60 – 80°C RPE – For analysis 2.5 L
Sprayer VidraFOC 712/1
Sodium sulphate anhydrous Merck 238597
Sulfuric acid 95-97% Merck 1007311000 CAUTION. Sulfuric acid 95-97%
TLC chamber Merck Z204226-1EA Rectangular TLC developing tanks, complete L × H × W 22 cm × 22 cm × 10 cm
TLC plate Merck 1057210001 TLC SilicaGel 60- 20×20 cm x 25 u
TLC Plate Heater CAMAG 223306 CAMAG TLC Plat Heater III
Toluene Carlo Erba 488551 CAUTION. Toluene RPE – For analysis – ISO – ACS – Reag.Ph.Eur. – Reag.USP 1 L
Vortex Fisher Scientific 10132562 IKA Agitador IKA vórtex 3
1-naphthol Sigma-Aldrich 102269427 CAUTION

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Thin Layer Chromatography-Based Separation of Mycolic Acid Variants: A Method to Separate Mycobacterial Cell Wall Lipids Based on Polarity. J. Vis. Exp. (Pending Publication), e21094, doi: (2023).

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