Protein extraction

Protein extraction

1. HeLa cells are grown in DMEM in 6 cm dishes at 37°C in a 5% CO2 incubator. 

2. Remove DMEM. 

3. Add 1 mL of ice-cold PBS. 

4. The cells are harvested using a scraper and centrifuged at 2,000 × g for 2 min at 4°C. 

5. Remove supernatants. 

6. Freeze cell pellets once at -30°C or -80°C. 

7. Solubilize the cell pellets with 120 uL of ice-cold Lysis Buffer (containing 0.2% DDM) for 20 minutes on ice. 

8. Divide the cell suspension into 90 uL and the remainder (~30 uL). 

9-1. Centrifuge the remainder (~30 uL) of the cell suspension at 10,000 × g for 2 min at 4°C. 

9-2. Determine the protein concentration of the supernatant fraction using a NanoDrop One spectrophotometer (Thermo Fisher Scientific). 

10. Add 10 uL of Lysis buffer containing Benzonase (Merck Millipore, 70664) to 90 uL of the cell suspension to give a final dilution of 1/200 of Benzonase. 

11. Add 100 uL of 2× SDS-PAGE sample buffer to the benzonase-treated cell suspension. 

12. The samples are denatured at 55°C for 15 min and then at 98°C for 5 min. 

13. Protein concentration is adjusted with 1× SDS-PAGE sample buffer (diluted with the same Lysis buffer). 

14. The samples (20 μg protein) are separated by SDS-PAGE. 

15. For in-gel fluorescence imaging, the gel is visualized with FUSION SOLO.7S.EDGE (Vilber-Lourmat) immediately after SDS-PAGE. 

15. For immunoblotting, proteins are transferred to Immobilon-P PVDF membranes (Millipore, WBKLS0500) using the Trans-Blot Turbo Transfer System (Bio-Rad). 

DMEM: Dulbecco’s Modified Eagle Medium (Sigma-Aldrich, D6546) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, 173012) and 2 mM Lglutamine (Gibco, 25030-081) 

DDM: n-dodecyl-β-D-maltoside (Nacalai Tesque, 14239-54) 

Lysis buffer: 25 mM HEPES-KOH (pH 7.2), 150 mM NaCl, 2 mM MgSO4, 0.2% DDM, and protease inhibitors (Sigma-Aldrich, P8340)