Centrifugation-Based Isolation of Chloroplast Inner and Outer Envelope Membranes

The chloroplast envelope contains an inner envelope and an outer envelope. They are structurally and functionally different, so they must be separated for study. The first method to purify the envelope was reported in the 1970s, including preparation of intact chloroplasts from Vidafaba tissue, destruction of the chloroplast by osmotic shock, and purification of the envelope by differential centrifugation. But the resulting preparation is an enriched encapsulated fraction, contaminated with a small but large amount of thylakoids, the inner chloroplast membrane. There are currently more than 15 envelope purification methods to improve the yield and purity of envelopes. Studies have shown that the inner membrane particle density of the outer membrane is lower than the inner membrane dose, indicating that the protein content of the outer membrane is lower. The outer membrane area of the chloroplast is larger than the inner membrane. Furthermore, the outer membrane is non-specifically permeable to low molecular weight compounds, whereas the inner membrane is impermeable to such compounds and contains several transport systems for metabolite transport. Therefore, the centrifugation method based on the light outer membrane and the heavy inner membrane can separate the inner and outer membranes.

Fig. 1. Diagram of isolation of the outer and inner envelope membranes of the spinach chloroplast envelope. (Block M A, 2018)

Services

A large number of enzymes related to photosynthesis are attached to the inner membrane of the chloroplast, and the protein content of the outer membrane is low. Therefore, centrifugation has become one of the common methods for separating chloroplast envelope. For many years, Lifeasible has devoted to developing multiple methods to isolate highly purified chloroplast envelopes, which have been well received by customers around the world. Our scientists continuously optimize the conditions of the centrifugation method, and have successfully obtained high-purity inner and outer envelopers from spinach and peas, which are suitable for functional and structural analysis of single chloroplast membranes. The process is as follows:

(1) Select highly purified chloroplasts to prepare membranes.

(2) Intact chloroplasts were isolated in isotonic medium. It is then cultured in hypertonic medium to induce chloroplast contraction and separation of the inner and outer membranes.

(3) Membranes are released from chloroplasts by exposure to moderately varying pressures.

(4) The chloroplast inner and outer membranes were separated by differential centrifugation.

(5) Determine the components of the envelope.

Competitive Advantages

• The inner and outer coatings with high purity can be obtained, which are suitable for subsequent analysis.
• The centrifugation method can avoid the destruction of inner membrane polypeptides during protease treatment, because some proteases may enter the inner membrane.
• The centrifugation method is easy to operate, and can obtain a complete envelope, which contains less adhesion areas of the outer membrane and the inner membrane.

Lifeasible can meet the needs of customers on time and on budget through a wide range of chloroplast inner and outer envelope membranes 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.

Reference

1. Block M A. (2018) Isolation of inner and outer membranes of the chloroplast envelope from spinach and pea[M]//Plastids. Humana Press. 137-144.