The integrity of the proteome of subcellular components such as the nucleus is largely determined by whether or not other cellular contaminants are removed from the isolated components during purification. Isolation of highly pure nuclei from plants is a difficult task. The source for this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillemment, M. Zivi, C. Damerweil, V. Mitchen, eds.

Operation method

nucleoprotein extraction

Materials and Instruments

Rice Suspension Cells
Homogenate Lysis buffer SDS Sample buffer Phosphate buffer

Move

( 1 ) Nuclei were isolated from rice suspension cells according to the method of Morre and Anderson, with some modifications in this paper. All steps for isolation of nuclei were carried out on ice or at 4°C. The nuclei were isolated from rice suspension cells according to the method of Morre and Anderson.

( 2 ) About 3 g of rice suspension cells were collected by centrifugation at 3000 g for 5 min and resuspended in 5 ml of pre-cooled PBS in an ice bath. The suspension is centrifuged again at 3000 g for 5 min. The precipitate is resuspended with 5 ml of homogenate and transferred to a glass mortar and ground to a homogenate.

( 3 ) The homogenate was filtered twice through a double layer of Miracloth (Calbiochem, Darmstadt, Germany).

( 4 ) The homogenate was transferred to a 15 ml falcon tube and centrifuged at 1000 g for 10 min.

( 5 ) The precipitate was gently resuspended with the homogenate and placed at the top of a 2.0 mol/L sucrose gradient and then centrifuged at 50,000 g for 30 min at 4°C. The sucrose gradient consisted of 37.5 mmol/L Tris-maleic acid (pH 6.5), 5 mmol/L MgCl₂, and 1% dextrin T500.

( 6 ) The supernatant was carefully removed and the precipitate was again gently resuspended with 5 ml of homogenate.

( 7 ) The resuspension was again placed at the top of a 2.0 mol/L sucrose gradient and centrifuged at 50,000 g for 30 min. The precipitate containing the nuclei was resuspended in 100 μl of homogenate and observed with a light microscope (Figure 9-1A). （The precipitate containing the nuclei was resuspended in 100 μl of homogenate and observed by light microscopy (Figure 9-1A).

( 8 ) The suspension containing nuclei is used to extract nuclear proteins. Protein samples for bidirectional electrophoresis can be extracted with lysis buffer or with SDS sample buffer for SDS polyacrylamide gel electrophoresis. Each 20 μl of nuclei in the suspension can be lysed in a glass homogenizer with 500 μl of Lysis Buffer or SDS Sample Buffer.



( 9 ) The purity of nuclear proteins is assayed by Western hybridization analysis using an anti-histone H1 antibody (SantaCruz Biotechnology, Santa Cruz, CA) (Fig. 9-1B).

( 10 ) Proteins extracted from purified nuclei ( 500 μg ) were separated using bidirectional electrophoresis with lysis buffer. (Figure 9-2 , see Note 1).

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