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Cat. No.: HCIDG-50 (for 10-50T)

Cat. No.: HCIDG-250 (for 50-250T)

Description

High-purity Chloroplast Isolation Kit by Density Gradient Centrifugation is a reagent kit designed to quickly extract intact, biologically active, high-purity chloroplasts from fresh plant leaves within one hour through grinding, filtering, centrifugation, and density gradient centrifugation. The chloroplasts obtained with this kit are of high purity and structural integrity, making them suitable for studies on electron transport and phosphorylation in the photosynthetic electron chain, transmembrane transport and assembly of chloroplast proteins, as well as in vitro chloroplast protein synthesis and localization. Additionally, the chloroplasts can be used as starting materials for the extraction of chloroplast proteins and the isolation and purification of chloroplast DNA and RNA.

Chloroplasts are double-membrane organelles unique to certain algae and higher plants that perform photosynthesis. They are involved in various metabolic processes, including amino acid synthesis, chlorophyll synthesis, and lipid biosynthesis[1]. In higher plants, chloroplasts are lens-shaped, with a diameter of about 5-10μm and a thickness of about 1-3μm. Typically, there are about 50-200 chloroplasts per mesophyll cell in higher plants, accounting for approximately 40% of the cell's cytoplasm. Although the majority of chloroplasts are found in leaves, they are also present in stems, seeds, and immature fruits.

This kit employs the method of density gradient centrifugation to isolate and purify high-purity, intact chloroplasts.

Components

• Chloroplast Extraction Buffer I
• Chloroplast Extraction Buffer II
• Density Gradient Separation Solution
• Cell Strainers

Features

• This kit has a wide range of applications. It is broadly applicable to various types of fresh plants, including monocotyledons (such as Epipremnum aureum, maize, wheat) and dicotyledons (such as Arabidopsis thaliana, tobacco, spinach, soybean, and cabbage). 
• The kit is easy to use, requiring no special disruption equipment, and can quickly obtain high-purity, intact, biologically active chloroplasts within one hour.
• The kit yields a high quantity of chloroplasts. Using this product, approximately 0.1-0.2 mg of intact chloroplasts can be obtained from each gram of leaves. For instance, with Epipremnum aureum, about 0.2 mg of high-purity, intact chloroplasts can be obtained from each gram of leaves using this product.
• Considering processing 0.2g or 1g of leaves per extraction, the small and medium packages of this kit can perform 10-50 and 50-250 extractions, respectively. The filters included in the kit are sufficient for only 10 and 50 extractions; for additional filters, it is recommended to purchase Cell Filters (70μm pore size, individually packaged, sterile).

Storage Conditions

Store at 4ºC, valid for 6 months. Chloroplast Extraction Buffer I and Chloroplast Extraction Buffer II should be stored at -20ºC, Density Gradient Separation Solution at 4ºC, and Cell Strainers at room temperature, valid for at least one year.

Precautions

• Acetone, ultrapure water or deionized water, scissors, centrifuge tubes, and disposable plastic pestles or glass homogenizers, mortars, and homogenizers are required. It is recommended to use Ultrapure Water (DNase/RNase-Free, Sterile).
• Chloroplasts are highly sensitive to temperature; therefore, all steps in the chloroplast isolation process must be performed on ice, and all solutions should be ice-cooled. Centrifugation should be carried out using a 4ºC centrifuge.
• During resuspension, if using standard pipette tips, cut off the tip to increase the diameter and gently pipette to avoid vigorous action that could rupture the chloroplasts.
• Throughout the experimental procedures, avoid vigorous shaking or pipetting. Handle gently and slowly to prevent chloroplast rupture.
• The chloroplast resuspension obtained is very prone to losing activity, so it is advisable to use it as soon as possible if performing activity assays.
• Thawed reagents must be completely thawed and mixed before use.
• If more cell filters are needed, it is recommended to purchase Cell Strainers (70μm pore size, individually packaged, sterile).
• This product is for scientific research use by professionals only. It is not intended for clinical diagnosis or treatment, nor for food or drug use, and should not be stored in residential areas.
• For your safety and health, please wear a lab coat and disposable gloves while handling.