Technical

Westernblot experimental protein extraction
source:Technical divisiondate:2022-05-11views:655

Adherent cell total protein extraction:

 

Rinse the adherent cells 2-3 times with TBS buffer, blotting the remaining liquid as much as possible the last time. Add an appropriate volume of cell total protein extraction reagent (add protease inhibitor within minutes before use) and lyse in the culture plate/flask for 3-5min. Shake the plate/flask repeatedly during this period to make full contact of the reagents with the cells. The cells and reagents were scraped off with a cell scraper and collected into a 1.5 mL centrifuge tube. Ice bath for 30 min, during which time, pipette repeatedly to ensure complete cell lysis. Centrifuge at 13000g for 5 min at 4°C and collect the supernatant, which is the total protein solution.

 

Total protein extraction from suspension cells:

 

The cells were collected by low-speed centrifugation, resuspended by adding an appropriate volume of cold PBS buffer, centrifuged at 2000 rpm for 10 min, and the supernatant was removed by suction. Repeat the above operation twice to collect the cell pellet. Add the appropriate volume of total cell protein extraction reagent (protease inhibitors are added within minutes before use) and shake. Ice bath for 30 min, during which time, pipette repeatedly to ensure complete cell lysis. Centrifuge at 13000g for 5 min at 4°C and collect the supernatant, which is the total protein solution.

 

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.

 

Cytoplasmic nuclear protein extraction:

 

Cells were harvested, resuspended in an appropriate volume of plasma protein extraction reagent (protease inhibitor was added within minutes before use), and vortexed for 15 seconds to completely suspend and disperse cells. If the cell pellet is not completely suspended and dispersed, the mixing time can be appropriately extended. Ice bath for 30 min. Mix at high speed for 5 seconds and centrifuge at 13000g for 10 minutes at 4°C. Pipette the supernatant into a pre-cooled centrifuge tube, which is the cytoplasmic protein. Aspirate the supernatant (try to remove the supernatant as much as possible to avoid contamination of plasma protein), add an appropriate volume of nucleoprotein extraction reagent (add protease inhibitor within a few minutes before use), and mix at high speed for 15 seconds to suspend and disperse the pellet completely. Ice bath for 30 min, shake vigorously for 10-20 s every 5 min, and centrifuge at 13000g at 4°C for 10 min. Pipette the supernatant into a pre-cooled centrifuge tube, which is the nuclear protein.