Arabidopsis total protein Preparation

Protocol from Extraction of total protein from Arabidopsis. Rob Larkin. CSH Protoc. 2007 Jan 1:2007:pdb.prot4680. doi: 10.1101/pdb.prot4680. A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. J F Martínez-García 1, E Monte, P H Quail. Plant J. 1999 Oct;20(2):251-7. doi: 10.1046/j.1365-313x.1999.00579.x.

Extraction method: TCA/acetone precipitation method

Protein extraction Procedure:

Grind Arabidopsis leaves thoroughly at low temperatures; Add twice the volume of Protein extraction buffer A(10% (w/v) TCA, 0.07% (v/v )β-ME, Acetone, Pre cooling at -20 ℃), vortex until the sample is fully mixed with the extract, and then place the sample at -40℃ overnight; After balancing the sample, centrifuge at 40000 g for 1 h at 4℃. Discard the supernatant and add 1 ml of Wash buffer (0.07% (v/v) β-ME, Acetone, Pre cooling at -20℃) to the precipitate to completely mix it with the wash buffer. Then, continuously add the wash buffer until the final volume is the same as Protein extraction buffer A from the previous step. Finally, place the sample in a -40℃ freezer for 1h; After balancing, centrifuge at 40000g for 1 h at 4℃, repeat 3 times; Discard the supernatant and perform vacuum drying on the precipitate.

Protein dissolution:

1. Weigh the protein powder and add Protein extraction buffer B (7M urea, 2M thiourea, 4%(w/v) CHAPS, 40mM DTT) (containing protease inhibitor) to the protein powder in a ratio of 0.1g protein to 3ml Protein extraction buffer B. Vortex the mixture until the protein powder and Protein extraction buffer B are thoroughly mixed and dissolved;

2. After complete dissolution of the protein, centrifuge at 4 ℃, 17940g for 1 h;

3. Transfer the supernatant to a new centrifuge tube, and complete the extraction of total protein.

Quantify the protein:

Determination of protein concentration using BCA method