The rapid development of proteomics in recent years has led us to a deeper understanding of protein function and interaction mechanisms. The key to the study of proteomics is protein extraction. This paper will introduce the extraction of total protein from adherent cells, suspended cells and tissues.

Extraction of total protein (TF) from adherent cells

Preparation of lysate (pH 8.5-9.0): 50mM Tris-HCl, 2mM EDTA, 100mM NaCl, 0.5%Triton X-100, adjuste the pH value to 8.5-9.0; add 100μg/mL lysozyme, 1μL/mL Protease inhibitor PMSF into the solution.

Without treatment of drugs:

(1) discard the culture medium carefully, and make the bottle upside down on the absorbent paper to absorb the culture medium;

(2) The cells were washed with 3mL of pre-cooled PBS (0.01M pH 7.2 to 7.3), and the washings were discarded, repeated three times then place the flask on ice;

(3) adding lysis solution, lysing on ice for 30min;

(4) cells were scraped to one side of the flask with a clean scraper, and then removed to the centrifuge tube with a pipette;

(5) centrifugation for 5min at 4°C and 12,000rpm;

(6) The supernatant was transferred to centrifuge tube and stored at -20°C.

With treatment of drugs:

Due to the impact of drugs, some cells would fall off into the solution, so cells in the culture medium should also be collected in addition to the above operation:

(1) pour the culture medium into the centrifuge tube, centrifugation for 5min at 2500rpm;

(2) discard the supernatant, add a certain amount of PBS and gently blow to wash it with a pipette, and then centrifugation for 5min, repeated once;

(3) discard the supernatant, add lysis solution and put it on the ice for 30min;

(4) mixed with the previous lysate, centrifugation for 5min at 4℃ and 12,000rpm, take the supernatant in the centrifuge tube and stored at -20 ℃.

Extraction of total protein from suspension cells

(1) the suspension cells were precipitated, centrifuged at 2500rpm for 10min, and the supernatant was discarded;

(2) adding a precool protein extraction reagent containing inhibitor;

(3) shake it for 15min gently;

(4) centrifugation at 14,000rpm for 15min, discard the precipitate, and transfer the supernatant into a new centrifuge tube immediately.

Extraction of total protein in tissues

(1) a small amount of tissue is placed in a 1~2mL homogenizer, cut the tissue into pieces as small as possible;

(2) add 400μL single detergent lysis solution in the homogenizer, homogenate, ice bath;

(3) make the organization as broken as possible;

(4) lysis for 30min, the lysate was transferred to a centrifuge tube, centrifuged at 12000rpm and 4℃ for 5min, and the supernatant was stored in centrifuge tube at -20℃.

Protein sample extraction is a prerequisite for subsequent analysis. The ideal method of protein separation should include all proteins as much as possible, non-polluting and the after-processing is simplified. Different system, different method, so we should choose the appropriate method to obtain the best analysis results.

Edited by Suzhou Yacoo Science Co., Ltd.