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 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…
Divulgaciones
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).