Isolation of Chloroplast Inner and Outer Envelope Membranes

Like many biofilms, the chloroplast envelope plays an important role in mediating the complex interactions between the chloroplast and the cytoplasm. The envelope is also the site of various biosynthetic reactions. Lifeasible's expertise in chloroplast engineering has allowed us to isolate the chloroplast envelope in the purified state by a variety of methods, a thorough study of the function and properties of the envelope. If you have any special requirements about our isolation of chloroplast inner and outer membranes services, please feel free to contact us. We are looking forward to working together with your attractive projects.

Chloroplast Inner and Outer Envelope Membranes

The chloroplast is surrounded by a pair of closely spaced membranes, the double-membrane envelope, consisting of an inner membrane surrounding the stroma or stroma and an outer membrane in contact with the cytoplasm. In addition to its structural role, the envelope mediates many important chloroplast functions. For example, chloroplast proteins synthesized on cytoplasmic ribosomes must pass through the envelope to reach their correct location. The envelope is also involved in various aspects of chloroplast biogenesis, including the formation of galactolipids, which are major components of the envelope and thylakoid membranes. The study found that the inner and outer membranes differed in structure and function. For example, the outer membrane exhibited a lower intramembrane particle density than the inner membrane dose, indicating that the outer membrane has a lower protein content. Furthermore, the outer membrane is non-specifically permeable to low molecular weight compounds, while the inner membrane is impermeable to such compounds and contains several transport systems for transporting metabolites. A thorough study of the function and properties of the envelope requires the ability to isolate it in a purified state.

Fig. 1. Representative scheme of chloroplast sub-compartments. (Bouchnak I, et al., 2018)

Services

Lifeasible is committed to developing methods to increase the yield and purity of the resulting membranes. Based on a modification of the method for isolating intact chloroplasts, including the ionic conditions used during chloroplast lysis, and the centrifugation protocol used to isolate disrupted chloroplasts. We can help you easily prepare a reasonable amount of envelopes (a mixture of the two), such as 5 mg of protein per kg of leaf, without contamination by thylakoids or other cell membranes. In addition, the structural features of the envelope hindered attempts to purify the inner and outer membranes separately during the course of the study. We attempted to isolate the chloroplast envelope component membranes to determine the properties of the individual membranes and study the function of the envelopes in more detail.

The chloroplast envelope is a relatively easy membrane system to isolate. On the one hand, it is abundant in plant cells, and studies have found that the chloroplast envelope area accounts for about 15% of the total membrane area. on the other hand, recent developments in chloroplast isolation procedures allow for the rapid purification of highly purified intact chloroplasts. This allows us to easily obtain the envelope through the uncontaminated chloroplast outer membrane. Our scientists have successfully developed several reliable methods for isolating the inner and outer membranes of chloroplasts, and we will make the best choice for you according to the characteristics of the different methods.

• Protease Treatment-based Isolation of Chloroplast Inner and Outer Envelope Membranes
• Centrifugation-based Isolation of Chloroplast Inner and Outer Envelope Membranes

Procedure for Separating the Inner and Outer Membranes of Chloroplasts

We have successfully prepared chloroplast envelopes from peas and spinach. Our services provide a molecular perspective on organelle movement and membrane lipid remodeling for plant survival under different environmental conditions. Ongoing and future studies of the chloroplast outer envelope will increase our knowledge of organelle biology and eukaryotic evolution. The general procedure for separating the inner and outer membranes of chloroplasts is as follows:

(1) Purification of intact chloroplasts.

(2) Purification and fractionation of chloroplast envelope membranes.

(3) Multiple ways to handle intact chloroplasts.

(4) Identification and quantitation of membrane polar lipids.

(5) Electron microscopy.

References

1. Bouchnak I, Moyet L, Salvi D, et al. (2018) Preparation of chloroplast sub-compartments from Arabidopsis for the analysis of protein localization by immunoblotting or proteomics[J]. JoVE (Journal of Visualized Experiments). 140: e58581.
2. Cline K, Andrews J, Mersey B, et al. (1981) Separation and characterization of inner and outer envelope membranes of pea chloroplasts[J]. Proceedings of the National Academy of Sciences. 78(6): 3595-3599.