Total protein Extraction from Arabidopsis

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 40,000 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 equal to the Protein Extraction Buffer A from the previous step. Finally, place the sample in a -40℃ freezer for 1h; after balancing, centrifuge at 40,000g 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 at 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 the protein is completely dissolved, centrifuge at 4 ℃, 17,940g for 1 h;

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

Quantify the protein:

Determine protein concentration by BCA method