Extraction and Purification of Plastoglobules from Photosynthetic Bacteria

Abstract

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

The video demonstrates the isolation of plastoglobules from cyanobacteria. It begins with a culture of cyanobacteria and proceeds through cell disruption using mechanical force. The release of photosynthetic pigments indicates successful lysis. A sucrose step gradient is then used to separate cellular components by density, allowing low-density plastoglobules to form a distinct band at the top. This fraction is collected and preserved for further analysis.

Protocol

1. Crude plastoglobule isolation

1. Crude plastoglobule extraction from cyanobacteria
   1. Grow a 50 mL culture of Synechocystis sp. PCC 6803 to the stationary phase (about 7-10 days) and adjust the cell density to an Optical Density at 750 nm (OD₇₅₀) of 2.0 using a spectrophotometer. For culture conditions, use BG-11 media and grow in an incubator at a light intensity of 150 µmol photons/m²/s, 2% CO₂, 32 °C, and with continuous shaking at 150 revolutions per minute (rpm).
   2. To remove the polysaccharides, wash the cells by centrifuging the 50 mL culture at 6,000 × g for....

Results

Figure 1: Isolation and immunoblotting of plastoglobules and thylakoids. (<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ascorbate	BDH	BDH9242
Bovine Serum Albumin	Proliant Biological	68700
French Pressure cell (model FA-079)	SLM/Aminco	N/A
HEPES	Sigma Aldrich	H3375
Magnesium Chloride	Sigma Aldrich	M8266
Multitron shaking incubator	Infors HT	N/A
Potassium Hydroxide	Fisher Chemicals	M16050
Reduced Cysteine	MP Biochemicals	101444
Sorbitol	Sigma Aldrich	S3889
Sucrose	Sigma Aldrich	S9378
Synechocystis sp. PCC 6803	N/A	N/A
Optima MAX-TL Ultracentrifuge	Beckman Coulter	A95761