Degradation of target protein in living cells by small-molecule proteolysis inducer

Bioorganic and Medicinal Chemistry Letters

Volumn 14, Issue 3, 2004, Pages 645-648

  Zhang, Dong ^a   Baek, Sun Hee ^a   Ho, Abby ^a   Kim, Kyungbo ^a  

^a University of Kentucky   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

UBIQUITIN;

ARTICLE; CELL ACTIVITY; CONTROLLED STUDY; HUMAN; HUMAN CELL; MOLECULE; PROTEIN DEGRADATION; PROTEIN TARGETING; TECHNIQUE;

EID: 1642575095     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2003.11.042     Document Type: Article

References (20)

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20. The incubated cells with SMPI at 37 °C were lysed by lysis buffer (20 mM Tris-HCl pH 7.4, 1% Triton X-100, 5 mM EDTA, 10 μL of Protease inhibitor and 860 μL of distilled water). The protein concentrations were measured using Bio-Rad protein assay reagent. SDS-PAGE was carried out with a 8% SDS separating gel. The separating gel (2.7 mL of 30% Acrylamide/Bis-, 2.5 mL of 1.5 M Tris pH 8.8, 4.6 mL of D.D.W., 50 μL of 20% SDS, 100 μL of 10% APS and 6 μL of TEMED) and stacking gel (1.7 mL of 30% Acrylamide/Bis-, 2.5 mL of 0.5 M Tris pH 6.8, 5.6 mL of distilled water, 50 μL of 20% SDS, 100 μL of 10% APS and 10 μL of TEMED) were prepared just before running. 20 mg of protein samples were loaded from each time points and control. The electrophoresis was performed at 120 V for 2 h using a running buffer system (3.03 g of Tris-Cl, 14.41 g of Glycin, 5 mL of 20% SDS, and distilled water up to 1 L). The proteins were then transferred to PVDF membrane (Amersham) at 250 mA electric current for 2 h using a transfer buffer system (3.78 g of Tris-Cl, 18 g of Clycine, 1.85 mL 20% SDS, and distilled water up to 1 L). The membranes were washed three times with PBST buffer, were then treated with 5% skim milk for 3 h at room temperature (or overnight at 4 °C). After 2 h-incubation with the primary MetAP-2 antibody (1:250 in BSA, Zymed) at room temperature (or overnight at 4°C), the membrane was incubated with the secondary antibody (1:10,000 in 3% skim milk, Amersham) at rt for 2 h. The membranes were then washed with PBST three times. Finally, the film was developed by Western blotting detection reagents (Amersham) using a Kodak film (Kodak X-OMAT AR).