Analysis of Heterocyst and Akinete Specific Glycolipids in Cyanobacteria Using Thin-layer Chromatography

Materials and Reagents

1. 50 mL of cyanobacterial liquid cultures containing heterocysts or akinetes

2. Pipette tips (10 µL, 200 µL, 1,000 µL) (LTS, RAININ, Mettler Toledo. Gießen)

3. 1.5 mL microcentrifuge tubes

4. 50 mL conical tubes

5. Soft graphite pencil

6. 30 cm ruler

7. Paper tape (masking tape)

8. Methanol ≥99.9% (HPLC grade) (Sigma, Merck, Darmstadt)

9. Chloroform (HPLC grade) (Roth, Karlsruhe)

10. Aluminium plate coated with silica gel (Macherey-Nagel, catalog number: 818033)

11. Acetic acid (Sigma, Merck, Darmstadt)

12. MilliQ water

13. 25% Sulfuric acid (H₂SO₄) (Carl Roth, catalog number: 0967.1)

Equipment

1. Pipettes (P1000, P200, P20 and P10) (RAININ, Mettler Toledo)

2. 100 mL Erlenmeyer flasks

3. 50 mL glass beaker

4. Centrifuges:

   Centrifuge (Eppendorf, 5417C) for microcentrifuge tubes

   Centrifuge (Eppendorf, Centrifuge 5804 R) for conical tubes

5. Vortex Genie^® 2 (Scientific Industries, Inc., catalog number: 6235684)

6. Fume hood

7. Hair dryer

8. Incubator or oven for TLC plate development (This can be any incubator that reaches the desired temperature of 180°C)

9. Flat-bed Scanner (e.g., EPSON Perfection V550 Photo)

10. Developing Chamber for TLC (Macherey-Nagel GmbH & Co. KG, catalog number: 9003500)

11. TLC sprayer with Erlenmeyer flask (e.g., DWK Life Sciences Kimble^TM Kontes^TM Reagent Sprayers Head Only)

Software

1. Scanner software (e.g., EPSON Scan)

Procedure

1. Cell culture

   The protocol requires cyanobacterial cultures grown in the presence of NO₃^- (filaments with vegetative cells only) and without NO₃^- (filaments containing heterocysts), or the cultures that were induced to form akinetes (Perez et al., 2016).

   
   

2. Thin-layer chromatography (TLC)

   Day 1. Sample preparation

   1. Take 3 × 1 mL of each culture and estimate the chlorophyll a concentration as described (Zavřel et al., 2015).

      Note: To obtain a good visible lipid pattern in the TLC plates, the sample should contain 20–50 µg Chl a.

   2. Centrifuge the estimated volume of culture at 3,000 × g and room temperature (RT) for 10 min (the cells were pelleted at equal Chl a amount in each sample).

   3. Add 30 mL of methanol:chloroform (1:1) to the pellet and mix well with a vortex mixer. This step will result in the extraction of lipids.

      Note: Always work under the fume hood when using organic solvents.

   4. Centrifuge at 3,000 × g for 10 min and transfer the supernatant to a 50 mL glass flask.

   5. Let it evaporate overnight under a fume hood. You may accelerate evaporation by applying a stream of nitrogen vapor. This would be necessary if detailed analysis is intended, such as precise identification and quantification of different glycolipid species by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS).

   
   Day 2

   1. Sample collection

      1. Add 200 µL of chloroform to the dried extract in the glass flask, dissolve the pellet by pipetting, collect the lipids and transfer to 1.5-mL microcentrifuge tubes. Repeat this step one more time to collect all the extract from the flask (This step should be done quickly because the chloroform evaporates very fast).

      2. Let the sample dry in the fume hood until a volume of 20–40 µL remains in the tube.

   2. Preparation of developing chamber

      1. Always prewash the chamber with methanol before doing an experiment.

      2. Mix the solvents (chloroform:methanol:acetic acid:water) in the chamber, at a ratio of 23:4:2.7:1 (170 mL of chloroform, 30 mL of methanol, 20 mL of acetic acid, and 7.4 mL of MilliQ water).

      3. Close the lid and seal the tank with paper tape. Do not move the chamber.

      4. Before using the solvent system (solvent mixture), equilibrate the chamber with it for 1 h.

   3. Spotting of samples and development of the TLC

      Note: This can be done while the chamber is being equilibrated.

      1. Using a soft pencil, mark the origin line (the line at which you will spot the samples) at 2 cm and the development line at 15 cm (predetermined solvent front height) from the bottom of the TLC plate.

         Notes:

         1. Always handle the TLC plate with gloves on. Never touch the surface of the plate even with gloves.

         2. Mark only the outer edges of the TLC plate, because the pencil may interfere with the samples and solvent.

      2. Use a micropipette (P20) to spot 5–10 µL of the samples on the origin line, at a distance of at least 1 cm from each other.

      3. After spotting, use a hair drier in low heat mode to dry the spots.

      4. Repeat steps b. and c. until the entire sample is used up and dried.

      5. Quickly place the TLC plate in the chamber to prevent solvent vapor elusion from the chamber.

      6. Let the samples run until they reach the development line (~1.5 h).

      7. Take the plate out from the chamber, and let it dry under the fume hood for 10–20 min (do not use a hair drier).

         Note: Always remove the remainder of the solvent from the chamber and transfer it to the organic waste.

      8. When the plate is completely dry, spray the whole plate with H₂SO₄ (25%) with a TLC sprayer, from a distance of 5–10 cm under the fume hood (spraying close to the plate can obfuscate the result).

         Note: Use lab apron, gloves, and surgical mask.

      9. Allow the plate to dry under the fume hood for a short time (2–5 min).

      10. Place the plate into an oven at 180°C for 60–120 s (until you see the bands).

      11. Use a flat-bed scanner to record the result (see Figure 2).

   Data analysis

   
   

   
   Figure 2. Thin-layer chromatogram of lipid extracts from different cell extracts of A. variabilis (1–3) and N. punctiforme (4–6). (1, 4) Vegetative filaments grown with nitrate. (2, 5) Heterocysts containing filaments grown without nitrate. (3) Akinetes induced by low light, or (6) phosphate starvation for 2 months. The blue arrow indicates one heterocyst-specific glycolipid (HG₂₆-diol). The red arrow indicates an unknown lipid in the akinete extract of A. variabilis. Figure adapted from Perez et al. (2018) .

   Acknowledgments

   This protocol is an adapted version of the method described by Nichols and Wood (1968) and Winkenbach et al. (1972).

   R.G. and R.P. were supported by the grant GRK1708 from the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation).

   Competing interests

   We declare no conflict of interests.

   References

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