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

Abstract

Source: Guallar-Garrido, S., et al.  Analysis 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…

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