Protein isolation from cultured cells

Protein isolation buffer

Protein isolation buffer should be made fresh from stock reagents when needed. The following buffer is ideal for isolating protein from cells for Western blotting. To make 10mL of buffer mix the following stock reagents:

1mL of 50% glycerol stock for a final concentration of 5%. (A 50% stock in water is easier to pipette compared to 100%).
1mL of 10% SDS stock for a final concentration of 1%. (Handle SDS powder in the fume hood or wear a face mask).
500uL of 200mM Tris stock pH 7.4 for a final concentration of 10mM.
Protease inhibitor. (Sigma-Aldrich has a range of protease inhibitors to consider).
Use deionised water to make a final protein isolation buffer volume of 10mL

Store stock reagents at room temperature.

Tris is used to maintain the pH. Glycerol helps maintain protein stability. SDS is a detergent that removes membrane lipids and denatures proteins. If you are not performing Western blotting and would not like to denature proteins, SDS can be replaced with 1mL of 10% Triton X-100 for a final concentration of 1%.

Protein isolation from cultured cells

Steps

1. Remove cell media and gently wash cells with phosphate buffered saline (PBS).

2. Add 200uL of protein isolation buffer for a 6 well plate or 100uL for a 12 well plate. May perform steps 1 & 2 in the tissue culture laminar flow cabinet then transfer plates on ice to the lab bench.

3. Scrape cells with a cell scraper and transfer to 1mL eppendorf tubes.

4. Place on ice for ten minutes.

5. Syringe slowly up and down 5 times with 22 (21 – 27) gauge needle to shear DNA. Syringe slowly to prevent foaming. (Larger gauge needles shear less but less debris clog the needle).

6. Centrifuge for 10 (5 – 20) minutes at 12000g at 4°C, then transfer protein containing supernatant to new tube and store at -20°C or -80°C.