Westernblot experiment protein extraction

1) Total protein extraction from adherent cells.

Wash the adherent cells with TBS buffer 2-3 times, aspirating the residue as much as possible on the last occasion. Add the appropriate volume of total cell protein extraction reagent (add protease inhibitor within a few minutes before use) and lyse in the culture plate/flask for 3-5 min, shaking the plate/flask repeatedly to make full contact between the reagent and cells. The cells and reagents are scraped off with a cell scraper and collected into a 1.5mL centrifuge tube. Ice bath for 30min, during which the cells are repeatedly puffed with a pipette to ensure complete lysis. centrifuge at 13000g for 5min at 4℃ and collect the supernatant, which is the total protein solution.

2) Total protein extraction from suspended cells.

The cells were collected by low-speed centrifugation, resuspended by adding an appropriate volume of cooled PBS buffer, centrifuged at 2000 rpm for 10 min, and the supernatant was aspirated. Repeat the above operation twice and collect the cell precipitate. Add the appropriate volume of Total Cellular Protein Extraction Reagent (add protease inhibitor within a few minutes prior to use) and shake. Ice bath for 30 min, during which the cells are repeatedly puffed with a pipette to ensure complete lysis. centrifuge at 13,000g for 5 min at 4°C and collect the supernatant, which is the total protein solution.

3) Total tissue protein extraction.

Tissue blocks were rinsed 2-3 times with pre-cooled PBS buffer to remove blood stains, cut into small pieces and placed in a homogenizer. Add 10 times the tissue volume of tissue protein extraction reagent (add protease inhibitors within minutes before use), and thoroughly homogenize in an ice bath. Transfer the homogenate to a centrifuge tube and shake. Ice bath for 30min, during which time, pipette repeatedly to ensure complete lysis of the homogenate. Centrifuge at 13000g for 5 min at 4°C and collect the supernatant, which is the total protein solution.

4)Cytoplasmic cytosolic protein extraction

Collect the cells, resuspend them in an appropriate volume of Plasma Protein Extraction Reagent (with protease inhibitor added within a few minutes prior to use), and shake and mix for 15 seconds to completely suspend and disperse the cells. If the cell precipitate is not completely suspended and dispersed, the shaking time can be extended appropriately. Shake for 5 seconds at high speed and centrifuge at 13,000g for 10 min at 4°C. Aspirate the supernatant into a pre-cooled centrifuge tube to obtain the cell pulp protein. Aspirate the supernatant (remove as much supernatant as possible to avoid contamination of the plasma protein), add the appropriate volume of nuclear protein extraction reagent (add protease inhibitor within a few minutes prior to use), and mix with high speed for 15 seconds to completely suspend and disperse the precipitate. The supernatant was aspirated into a pre-chilled centrifuge tube and the nucleoprotein was extracted.