Cellular Protein Extraction Reagent

Cellular Protein Extraction Reagent 
 
DESCRIPTION: 
This reagent warrants highly efficient total protein extraction from cultured mammalian cells. 
The extracted proteins are compatible with Bradford and BCA protein assays or SDS-PAGE. 
The reagent is suitable for protein extractions from adherent cells grown in flasks or 6/12/24/96-well plates. 
The reagent is equally suitable for protein extractions from cells in suspension. 
Protein extraction can be completed in 5 minutes. 
Mechanical disruption of cells (by freeze/thaw or sonication) is not required. 
The reagent does not contain protease inhibitors. 
A culture of a million (106) cells is expected to weigh approximately 5 mg and yield approximately 0.3 mg protein, depending on the cell type.   
 
THE PROTOCOL FOR PROTEIN EXTRACTION FROM ADHERENT CELL MONOLAYERS: 
Decant the culture medium from the flasks/plates. 
Wash the flasks/wells once with warm (37°C) 1X PBS, pH 7.4. 
Add the Cellular Protein Extraction Reagent to the flasks/wells. The appropriate reagent amounts are shown in the Table below.          
 
Plate size / surface area          Suggested reagent volume
        100 mm                                          1000 µL
         60 mm                                             500 µL
          6-well                                         400 µL per well
         12-well                                        200 µL per well
         24-well                                        100 µL per well
 
Shake the flask/plate gently for 5 minutes at room temperature. 
Transfer the lysate to a microcentrifuge tube.  
Centrifuge the lysate at approximately 13,000 × g for 5-10 minutes to pellet the cell debris.  
Transfer the supernatant to a new tube for analysis and/or assays. 
 
THE PROTOCOL FOR PROTEIN EXTRACTION FROM CELLS IN SUSPECSION: 
Pellet the cells by centrifugation at 3000 x g for 10 minutes. 
Wash the cells by resuspending in approximately 1 mL of warm (37°C) 1X PBS, pH 7.4. Pellet the cells by centrifugation at 3000 x g for 10 minutes. 
Add an appropriate amount of Cellular Protein Extraction Reagent to the cells. A culture of 2x106 cells is expected to weigh 10 mg. At least 100 µL of reagent is required for each 10 mg of cells. 
Shake the mixture for 10 minutes at room temperature. 
Centrifuge the lysate at approximately 13,000 × g for 5-10 minutes to pellet the cell debris.  
Transfer the supernatant to a new tube for analysis and/or assays