Materials and Reagents

EDTA free protease inhibitors (F. Hoffmann-La Roche, catalog number: 05892988001 )
Proteasome inhibitor MG-132 (Calbiochem^®, catalog number: 474790-5MG )
Phosphate buffered saline (PBS)
Tris
Ni-NTA-Agarose beads (QIAGEN, catalog number: 30210 )
Urea
Guanidinium-HCl ( Sigma-Aldrich, catalog number: G4505-25G )
Imidazole (Sigma-Aldrich, catalog number: 12399 )
Triton X-100
Glycerol
β-mercaptoethanol
SDS
Bromophenol blue
Lysis buffer (see Recipes)
Wash buffer (see Recipes)
Elution buffer (see Recipes)
4x laemmeli buffer (see Recipes)

Equipment

Centrifuges
Sonicator
Western blotting equipment
Tissue culture plates
1.5 ml eppendorf tube
15 ml falcon tube

Procedure

Seed 1 x 10⁶ cells on 10 cm tissue culture plates.
After 8-12 h (around 40% of confluency) cells were transfect with 0.8 μg of plasmid for the His tag protein.
4 h before stop the culture add proteasome inhibitor MG-132 to a final concentration of 25 μM.
48 h after transfection aspirate the medium and wash the cells twice with pre-chilled PBS (cell can be keeping freezing on the plates at -80 °C until the day of the purification).
Scrape cell with 1 ml of lysis buffer and transfer it in 1.5 ml eppendorf tube.
Sonicate cells on ice twice for 10 sec with 1 min break (the sonicator probe should go under the lysate surface to avoid foaming but taking care do not touch the walls of the eppendorf tube).
Spin cells 10 min at 11,000 rpm at 4 °C.
Transfer the supernant in to 15 ml falcon tube and add 4 ml more of lysis buffer.
Add 75 μl of Ni²⁺-NTA-agarose beads pre-equilibrate with lysis buffer (take 150 μl of Ni²⁺-NTA-agarose bead and spin 2 min at 4,000 rpm, discard the supernant and wash with 150 μl of lysis buffer four times, last time add 75 μl of lysis buffer and use).
Incubate for 4 h at RT with gently agitation.
Wash the beads at RT incubating 5 min with the following buffers:
1. Once in lysis buffer.
2. Once in wash buffer.
3. Twice in wash buffer plus 0.1% Triton X-100 (spin 2 min at 4,000 rpm each time to recover the beads).
Elute products by incubating beads in 75 μl of elution buffer for 20 min at RT with gentle agitation.
Spin the beads and transfer supernant in to an eppendorf.
Add 4x laemmeli buffer and boil for western blot analysis.

Recipes

Lysis buffer
6 M guanidinium-HCl
0.1 M Na₂HPO₄/NaH₂PO₄
10 mM Tris-HCl (pH 8)
0.005 M imidazol
0.01 M β-mercaptoethanol
Wash Buffer
8 M urea
0.1 M Na₂HPO₄/NaH₂PO₄
10 mM Tris-HCl (pH 6.8)
0.005 M imidazol
0.01 M β-mercaptoethanol
Elution buffer
0.2 M imidazol
0.15 M Tris-HCl (pH 6.8)
30% glycerol
0.72 M β-mercaptoethanol
5% SDS
4x laemmeli buffer
2 ml 1 M Tris-HCl (pH 6.8)
4.0 ml 20% (w/v) SDS
4.0 ml glycerol
4 mg bromophenol blue
0.2 ml 14.3 M β-mercaptoethanol

Acknowledgments

This protocol was adapted from Gajjar et al. (2012). This work was supported by INSERM, La Ligue Contre le Cancer, and RECAMO CZ.1.05/2.1.00/03.0101. M.M.C. was supported by JSPS, AXA Research Fund, and Fundacao para a Ciencia e Tecnologia of Portugal. M.G. was supported by a research fellowship from Indo-French Centre for the Promotion of Advanced Research (IFCPAR) and from the ARC.

References

Gajjar, M., Candeias, M. M., Malbert-Colas, L., Mazars, A., Fujita, J., Olivares-Illana, V. and Fahraeus, R. (2012). The p53 mRNA-Mdm2 interaction controls Mdm2 nuclear trafficking and is required for p53 activation following DNA damage. Cancer Cell 21(1): 25-35.
Xirodimas, D., Saville, M. K., Edling, C., Lane, D. P. and Lain, S. (2001). Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 in vivo. Oncogene 20(36): 4972-4983.