Chloroplast Isolation from Spinach Leaves

Chloroplasts are plant-specific organelles. They are not only the site of photosynthesis, but also of many other important metabolic pathways, such as the synthesis of amino acids, vitamins, lipids and pigments. Currently biochemical, physiological and proteomic analysis of chloroplasts can only be achieved by using pure and intact chloroplasts that preserve metabolic activity and functions around the envelope. However, the chloroplast is an extremely fragile structure. Therefore, techniques and expertise are required to avoid large-scale rupture of the two envelopes during chloroplast isolation. Spinach leaves have important advantages in basic research of genetics and molecular biology, and are widely used as model organisms in plant biology. Available almost permanently on the market, spinach also contains low levels of phenols and other inhibitory compounds (ubiquitous in many cultivated species), making it an excellent choice for easily obtaining fully competent CO₂-fixing chloroplasts.

Fig. 1. Extraction of membrane proteins from spinach chloroplast thylakoids. (Korotych O, et al., 2019)

Services

Spinach, readily available all year round, remains the mode of choice for large-scale production of pure chloroplasts with high integrity. For many years, Lifeasible has been working on the independent isolation of chloroplasts from different plant tissues. We have developed a convenient protocol for the purification of chloroplasts from spinach leaves. Our scientists can isolate intact chloroplasts and other organelles from spinach leaf protoplasts, assisting you in studying the nature of the inter-organelle relationships for photorespiration. Additionally, we provide a rapid method to isolate intact chloroplasts from spinach leaves. The key to this method is to obtain a chloroplast-rich suspension (crude chloroplast extract) by differential centrifugation and to recover pure and intact chloroplasts using Percoll density gradient centrifugation. The chloroplasts isolated by this method have been successfully applied to study the photosynthetic electron transport mechanism. The basic process of spinach chloroplast separation is as follows:

(1) Preparing spinach leaves and homogenize the tissue.

(2) Centrifuge the homogenate.

(3) Purifing the chloroplasts by isopycnic centrifugation.

(4) Determination of chlorophyll content.

(5) Evaluation of chloroplast integrity.

Competitive Advantages

• With a high degree of integrity.
• Available in large quantities from the market all year round, no plant growing facilities are required.
• Spinach leaf protoplasts are of great value in the study of photosynthesis and photorespiration mechanisms.
• Homogeneous "naked leaf cell" preparations can be manipulated for studies.
• The procedure is fast and requires only two short centrifugation steps.
• The obtained intact chloroplasts are suitable for the study of photosynthetic electron transport.
• Chloroplasts isolated from spinach leaves were less fragile than those isolated from Arabidopsis leaves.

Lifeasible can meet the needs of customers on time and on budget through a wide range of chloroplast isolation strategies. Our aim is to be customer-centric and to provide the highest quality service to customers around the world. Our skilled and dedicated scientific researchers ensure that the most appropriate methods and techniques are selected for each specialized chloroplast project. Our customer service representatives are enthusiastic and trustworthy 24 hours a day, 7 days a week. If you are interested in our services, please feel free to contact us for more information or a detailed discussion.

References

1. Korotych O, Mondal J, Gattás-Asfura K M, et al. (2019) Evaluation of commercially available styrene-co-maleic acid polymers for the extraction of membrane proteins from spinach chloroplast thylakoids[J]. European Polymer Journal. 114: 485-500.
2. Kubis S E, Lilley K S, Jarvis P. (2008) Isolation and preparation of chloroplasts from Arabidopsis thaliana plants[M]//2D PAGE: Sample Preparation and Fractionation. Humana Press. 171-186.