Density Gradient Purification of Cyanobacterial Plastoglobules

Abstract

Source: Shivaiah, K., et al. Plastoglobule Lipid Droplet Isolation from Plant Leaf Tissue and Cyanobacteria. J. Vis. Exp. (2022).

This video demonstrates the isolation of plastoglobules from cyanobacteria using sucrose density gradient centrifugation, highlighting their lipid-rich, low-density nature and ensuring their structural preservation for downstream analysis.

Protocol

1. Harvesting pure plastoglobules

1. Cyanobacteria processing
   1. Produce a sucrose gradient in four 2.5 mL ultracentrifuge tubes layered first with 500 µL of the crude plastoglobules from step 1.3 mixed with 750 µL of medium R 0.7, then overlay with 750 µL of medium R 0.2 (Table 1)
   2. Centrifuge the sucrose gradient at 150,000 x g for 90 min at 4 °C. Collect the pure plastoglobules (Figure 1C) with a syringe and 22 G needle from the top phase of the sucrose gradient and transfer to a 1.5 mL tube.
   3. Aliquot the pure plastoglobules and flash-freeze in liquid nitrogen. Store directly at −80 °C or lyophilize to a dry powder.

Table 1: Buffer recipes for plastoglobule isolation from cyanobacteria.

Medium R	50 mM HEPES-KOH (pH 8.0)
	5 mM MgCl2
Medium R 0.2	50 mM HEPES-KOH (pH 8.0)
	5 mM MgCl2
	0.2 M sucrose
Medium R 0.7	50 mM HEPES-KOH (pH 8.0)
	5 mM MgCl2
	0.7 M sucrose

Results

Figure 1: Isolation and immunoblotting of plastoglobules and thylakoids. (A) A re...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
HEPES	Sigma Aldrich	H3375
Magnesium Chloride	Sigma Aldrich	M8266
Sucrose	Sigma Aldrich	S9378
Synechocystis sp. PCC 6803	N/A	N/A
Optima MAX-TL Ultracentrifuge	Beckman Coulter	A95761