Root meristem cell nuclear protein extraction experiment
The isolation and extraction of nuclear proteins has facilitated the study of plant proteomics. This technique relies on subcellular isolation with which the proteome of a subcellular organelle can be identified, and this technique is also used for the nucleus. The nuclear protein isolation method is based on the different solubility of proteins in buffers of different ionic strengths. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillmant, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Root meristem cell nuclear protein extraction
Materials and Instruments
Nucleus Move 3.1 Plant material For more product details, please visit Aladdin Scientific website.
Ficoll Glucosamine Sulfate Tris-HCl EDTA Octanol
Gum Arabic High Speed Dispersing Mixer
( 1 ) Onion bulbs, with the brown dry outer skin removed, are washed in tap water and each placed upright in a drum-shaped glass container filled with about 90 ml of filtered water so that only the bottom is submerged (see Note 3). The water must be aerated at a rate of 10-20 ml/min. The water must be changed every 24 h (see Note 4).
( 2 ) After 2-3 days of incubation, most bulbs will have grown many roots; after 3 days of incubation, 95% of the scales can be used to grow more than 40 roots per stem, each at least 2 cm long. these roots can be used for collection.
( 3 ) Using a scalpel or razor blade, remove the crown of the root and then clip the first 3 mm of the root tip with forceps; this is the root meristem (see Notes 5 and 6). Take approximately 500 mg of phloem tissue.
( 4 ) Place the sample in a 4 cm diameter covered petri dish containing 6 ml of extraction medium, with the protease inhibitor PMSF added before use.
( 5 ) Collect a total of 1 g of root meristem.
( 6 ) The petri dish must be placed on ice.
( 7 ) Note: The experiment must be performed in a fume hood.
( 8 ) Use a vacuum pump to remove gas from the sample solution for 10 min to promote penetration of the medium into the sample.
3.2 Purification of nuclei
( 1 ) Root meristematic tissues were placed in the extraction medium (which can be left at 4°C for 15 min to overnight) and filtered through a mesh of 100 μm, 50 μm, and 30 μm in diameter, respectively.
( 2 ) A spatula is used to collect the residual sample left on the nylon cloth into the extraction medium and the process of filtration is repeated twice. The samples in the tubes were stirred during homogenization using a stirrer rotating bar at a speed of 16,000 r/min for 3 times for 5 s each, with a pause of 5 s between the two times.
( 3 ) The homogenized filtrate was collected and the filtrate was rinsed by centrifugation at 800 g at 4°C for 10 min.
( 4 ) Re-dissolve the precipitate in 1 ml of extraction medium. The solubilization is aided by suction from a lance tip and centrifuged more than twice. At the end of this process, the precipitate contains purer nuclei.
( 5 ) Transfer the precipitate into a 1.5 ml centrifuge tube containing 1 ml NSB. The nucleus fraction is identified under a light microscope and examined for abundance and purity. Phase contrast microscopy can be used, but simple stains such as methyl green/piperonin, toluidine blue, and 4, 6- diamidino-2-phenylindole (DAPI) can help to identify nuclei (see Note 8 and Note 9).
3.3 Protein extraction by nucleus fractionation
We designed this set of experiments using the method described by Penman and co-workers [ 10], with appropriate modifications [14, 15]. The most important modification was the introduction of a step to extract most of the soluble proteins. Nuclear proteins that are reported to be soluble in low ionic strength buffers are functional because they are enriched in ribonucleoproteins that are highly active in nuclear RNA metabolism [8, 9, 16, 17].
Overall, this method consists of a series of protein fractionation methods using solubility as a criterion. The sample solution increases rigor and ionic strength in turn. The steps of the extraction method are described below.
1. Fractionation of membranes and residual cytoskeleton
( 1 ) The first step is the reaction of purified nuclei in NSB, a buffer enriched with descaling agents.
( 2 ) ( V/V ) NP-40 and 0.5% ( V/V ) sodium deoxycholate were added to the nuclei sample solution and shaken in a shaker for 10 min at 4°C. The tubes were then shaken in a vortex oscillator 2 times for 20 s each time.
( 3 ) Centrifuge the sample at 1000 g for 10 min at 4°C and collect the supernatant, which is the first fraction (membrane and residual cytoskeleton, Figure 8-1, lane M).
2. Soluble fraction (S2)
The precipitate is re-dissolved on a vortex shaker with 1 ml of low ionic strength buffer. The precipitate is shaken in the shaker at 4°C for 1 h, then centrifuged at 1000 g for 10 min at 4°C. The supernatant is the second fraction, called the S2 extract, which contains ribonucleoproteins (Figure 8-1, S2).
3. Chromatin fraction
( 1 ) Dissolve the precipitate in a vortex shaker with 400 μl of DNAase I containing 100 μg/ml RNAase-free DNAase and 0.5% Triton X-100 NBS. Shake at room temperature for 30 min, which will enzymatically digest the DNA.
( 2 ) Add ammonium sulfate (400 μl) to extract the digested DNA and react for 5 min at room temperature (see Note 10). As in the previous steps, the reaction is done in a rotating tracked oscillator.
( 3 ) Centrifuge at 2000 g for 10 min and remove the supernatant; the fraction contains proteins associated with chromatin (Figure 8-1, Chr).
The proteins are diluted before remeasurement and the final result is calculated based on the number of dilutions.