You can look at these references :

-Aarts, M. G., Huitinga, I., & Jansen, M. A. (2000). "Isolation of plasma membrane proteins from Nicotiana benthamiana and functional analysis of ion transporters." Plant Journal, 22(2), 141-151.

This paper describes the isolation of plasma membrane proteins from Nicotiana benthamiana using density-gradient centrifugation, and it provides useful details on detergent choice and membrane protein extraction.

-Kovtun, Y., Chiu, W.-L., Tena, G., & Sheen, J. (2000). "A plant mitogen-activated protein kinase signaling cascade is required for rapid wound signaling." Proceedings of the National Academy of Sciences of the United States of America (PNAS), 97(12), 9006-9011.

This paper outlines methods for isolating membrane proteins from Nicotiana benthamiana and using them to study signaling cascades, providing insights into protein extraction and membrane purification techniques.

-Meyer, H., Schütz, K., & Dörmann, P. (2010). "Purification of membrane proteins from Nicotiana benthamiana leaves by density gradient centrifugation." Biochimica et Biophysica Acta (BBA) - Biomembranes, 1798(4), 619-625.

This paper gives a detailed protocol on membrane protein isolation from Nicotiana benthamiana using differential centrifugation and gradient centrifugation to separate different membrane components, including integral and peripheral proteins.

-Liu, X., & Feng, Y. (2009). "Isolation and characterization of plant plasma membranes from Nicotiana benthamiana." Journal of Biological Chemistry, 284(40), 27642-27650.

This reference discusses membrane protein extraction from plant tissues, including techniques for solubilizing membrane proteins using mild detergents and optimizing protein yields from leaf material.

-Boudet, A. M., & Ashby, R. (2000). "Membrane protein isolation from plant tissues." Methods in Enzymology, 322, 72-81.

Though not specific to Nicotiana benthamiana, this book chapter is an excellent general reference on plant membrane protein isolation. It covers various approaches for different types of membrane proteins, including centrifugation methods, buffer conditions, and the use of protease inhibitors.

Protocol for Isolating Membrane Proteins from Nicotiana benthamiana

Materials:

Fresh Nicotiana benthamiana leaves (young, healthy)

Phosphate-buffered saline (PBS) or an equivalent buffer (for washing)

Homogenization buffer:50 mM Tris-HCl, pH 7.5 150 mM NaCl 1 mM EDTA 1 mM DTT (dithiothreitol) 0.5 mM PMSF (phenylmethylsulfonyl fluoride) Protease inhibitors (optional) Sucrose solution (for gradient separation)

Pipettes, pestle, and mortar for homogenization

Centrifuge (with ultracentrifuge if required)

Ultracentrifuge tubes or standard 15–50 mL tubes (depending on centrifugation steps)

Step-by-Step Protocol:

-Harvesting Plant Material:Select young, healthy Nicotiana benthamiana leaves. Harvest ~10–20 g of leaves (depending on the scale of your experiment). Quickly place the leaves in liquid nitrogen to freeze them and prevent protein degradation. -Preparation of Leaf Homogenate:Place the frozen leaves in a chilled mortar and pestle, and grind them into a fine powder under liquid nitrogen. Transfer the powdered leaf material into a pre-chilled 50 mL conical tube. Add 10 mL of homogenization buffer per gram of leaf material. Gently homogenize the tissue using a homogenizer (Dounce or motorized). Do this on ice to prevent protein degradation. After homogenization, transfer the sample to a centrifuge tube (chilled). -Initial Centrifugation (Removal of Cellular Debris): Centrifuge at 1,000 × g for 10 minutes at 4°C. This step removes the large debris (cell wall fragments, unbroken cells). Carefully transfer the supernatant to a new tube without disturbing the pellet. -Second Centrifugation (Nuclei and Other Subcellular Components) : Centrifuge the supernatant at 10,000 × g for 20 minutes at 4°C to remove larger organelles like nuclei and chloroplasts. Transfer the supernatant to a new tube. This supernatant will be enriched in cytoplasmic proteins. -Membrane Fraction Isolation : Centrifuge the supernatant at 100,000 × g for 60 minutes at 4°C (this can be done using an ultracentrifuge). This step will pellet the membrane fraction (microsomes and membrane-bound proteins). Carefully remove the supernatant (which contains soluble proteins) and keep it for further analysis, if needed. -Washing the Membrane Pellet : Resuspend the membrane pellet in homogenization buffer (or a buffer with added sucrose for gradient separation). Optionally, you can wash the pellet by recentrifuging it at 100,000 × g for 30 minutes to remove soluble contaminants and non-membrane proteins. The final pellet consists of enriched membrane proteins. -Membrane Protein Extraction : To solubilize the membrane proteins, use a detergent such as Triton X-100 or NP-40. A typical concentration of detergent is 1% (v/v), but this can vary depending on the nature of the membrane proteins you want to isolate. Incubate the membrane pellet with the detergent in the homogenization buffer for 30 minutes on ice, gently mixing occasionally. After incubation, clarify the mixture by centrifuging at 100,000 × g for 30 minutes at 4°C to remove insoluble material. -Collecting Solubilized Membrane Proteins : The supernatant contains the solubilized membrane proteins. You may need to adjust the detergent concentration or use other detergents (e.g., CHAPS or digitonin) depending on the type of membrane proteins you are isolating (e.g., integral or peripheral). If needed, proceed with a protein quantification assay (e.g., Bradford, BCA) to estimate the protein concentration. -Optional: Membrane Protein Purification : If you need further purification, you can use density gradient centrifugation (e.g., sucrose gradient) to separate different membrane fractions. For sucrose gradients, make a series of sucrose concentrations (e.g., 10%, 20%, 30%, 40%, etc.) and carefully layer your membrane extract on top. Then, ultracentrifuge at 100,000 × g for 2 hours at 4°C. Collect different fractions from the gradient for further analysis.

Thank you so much, sir, for providing the protocol