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Plant Proteomics

Chloroplast Iso Kit


Description:

The Chloroplast Isolation Kit provides a quick and efficient procedure to isolate intact chloroplasts from plant leaves. Intact chloroplasts are the best starting material for studies of chloroplastic processes such as carbon assimilation, electron flow and phosphorylation, metabolic transport, or protein targeting. The chloroplast fraction can be further extracted to obtain membrane, stroma, or thylakoid proteins as well as chloroplastic DNA and RNA.

The chloroplast isolation method includes mechanical cell wall and membrane breakage, removal of cell debris and unbroken leaf tissue by filtration, collection of total cell chloroplasts by centrifugation, and separation of intact from broken chloroplasts using a Percoll® layer or gradient. The Chloroplast Isolation Kit has been tested for use with spinach, pea, lettuce, cabbage, mangold, and tobacco.

 
Kit Components:
  • Chloroplast Isolation Buffer 5x (CIB)
  • Percoll
  • Bovine Serum Albumin (BSA)
  • Filter Mesh 100


Ferricyanide photoreduction - a measure of intact chloroplasts
This assay is based upon the inability of the ferricyanide to cross the chloroplast envelope and to react with the electron transport system in the thylakoid membranes. Ferricyanide reduction, as indicated by the decrease in the absorbancy at 410 nm, occurs only when ruptured chloroplasts are present in the preparation.
The degree of integrity of the chloroplast preparation is assessed by comparing the rate of ferricyanide reduction upon illumination before and after osmotic shock of the chloroplasts.
Analysis of the results, presented here, indicates that 88% of the chloroplasts in the spinach chloroplast preparation are intact.
Chloroplasts (equivalent to 25 µg/ml chlorophyll) prepared using CP-ISO were illuminated in the presence of 1.5 mM ferricyanide. The reduction of ferricyanide was measured spectrophotometrically (410 nm).
Graph A demonstrates the change in absorbancy of the two samples (before and after osmotic shock) during 6 minutes.
Graph B shows bars representing the slopes of the lines in Graph A.

Ordering Information

Product Product Name Package Size Technical Bulletin
CP-ISO Chloroplast Isolation Kit 1 Kit X