Isolation of Chloroplast Thylakoid Lumen

The thylakoid lumen of the chloroplast is an important site for oxygen release, plastocyanin-mediated electron transfer, and photoprotection. Furthermore, luminal proteins play important roles in regulating thylakoid biogenesis and the activity and turnover of photosynthetic protein complexes. There is an urgent desire to delve deeper into chloroplast thylakoid lumen proteins. Lifeasible's expertise in chloroplast engineering has allowed us to use advanced isolation methods to obtain high yields of pure thylakoid lumens for various proteomic analyses, immunoassays, enzyme activity assays, and protein complex studies. If you have any special requirements about our isolation of chloroplast thylakoid lumen services, please feel free to contact us. We are looking forward to working together with your attractive projects.

Chloroplast Thylakoid Lumen

The chloroplast contains six protein-containing compartments, the outer envelope, outer envelope space, inner envelope, stroma, thylakoid membrane, and thylakoid lumen. The study found that proteins in these compartments are closely related to the function of the chloroplast. Currently, each compartment's proteome presents its own challenges. For example, in the stroma there are dynamic range problems due to the high abundance of ribulose bisphosphate carboxylase. In the thylakoid lumen, luminal proteins are less abundant compared to other chloroplast proteins. The study found that the inner surface of the thylakoid membrane wraps a narrow, continuous cavity, the thylakoid lumen. In addition, the researchers observed spinach thylakoids through electron microscopy and found that the chamber is a dense space. To date, there are few methods used to isolate thylakoid lumens. Moreover, the knowledge of the cavity from a compositional and functional perspective is fragmented, collected from several independent methods, only for a single aspect of this cavity. Therefore, scientists worked to develop a technique to obtain the thylakoid lumen from the inside out to study the proteome.

Fig. 1. The majority of thylakoid lumen proteins with experimentally verified roles are involved in the function of either the PSII complex or the PSI–NDH supercomplex. (Järvi S, et al., 2013)

Services

The thylakoid lumen is a continuous extracellular space that is poorly characterized compared to other chloroplast lumens. Lifeasible is committed to developing reliable methods to isolate chloroplast thylakoid lumen with high purity and high yield. In addition, we characterized the thylakoid lumen containing high concentrations of proteins. Here, we can help you obtain thylakoid luminal proteomes in high purity form to study the function of luminal proteins. At the same time, the isolated chloroplast thylakoid lumens can be used to study photosynthetic electron transfer and ultimately plant fitness. We have successfully isolated luminal fractions from spinach, Arabidopsis, and peas by disrupting isolated thylakoid membranes. The process is as follows:

(1) Intact chloroplasts were isolated from leaf material by homogenization and differential centrifugation.

(2) Thylakoid membranes were obtained by fragmenting chloroplasts by osmotic shock, and washed several times to remove contaminating chloroplast matrix proteins and peripheral membrane proteins.

(3) Soluble thylakoid luminal proteins were isolated by crushing the separated thylakoid membranes by Yeda.

(4) Measure chlorophyll, protein and oxygen release.

(5) Electrophoresis and Western blot analysis.

(6) Enzyme activity assay.

Competitive Advantages

• The abundance of thylakoid luminal proteins is rather low compared to matrix and thylakoid proteins, so a large amount of leaf material (usually 60–120 g) is required to obtain usable thylakoid luminal fractions for proteomic analysis.
• Fast, reliable, and easy-to-operate method that can be prepared within 1 day.
• A variety of luminal proteins were obtained and characterized.

References

1. Järvi S, Gollan P J, Aro E M. (2013) Understanding the roles of the thylakoid lumen in photosynthesis regulation[J]. Frontiers in plant science. 4: 434.
2. Hall M, Mishra Y, Schröder W P. (2011) Preparation of stroma, thylakoid membrane, and lumen fractions from Arabidopsis thaliana chloroplasts for proteomic analysis[M]//Chloroplast Research in Arabidopsis. Humana Press, Totowa, NJ. 207-222.