Phosphate buffer

Example 8

The pET-14B vector containing a His6-tagged Mdm2_25-117 fusion protein was transformed into BL21(DE3) competent E. coli (Novagen) in M9 minimal media with ¹⁵NH₄Cl as the main nitrogen source. Protein production was induced with 0.4 mM IPTG at O.D.600 and incubated for 16 hours at 15° C. Bacteria were harvested and resuspended in the lysis buffer with 20 mM Phosphate buffer (Research Products International, Corp.), 100 μM DTT (Fisher), 1 mM EDTA (Sigma), 0.5% TritonX 100 (Sigma), 1 mg/mL Pepstatin A (Research Products International, Corp.), 10 mg/mL Leupeptin A (Research Products International, Corp.), 500 μM PMSF (sigma), and 0.5% glycerol at pH 8.0. Pellets were lysed by sonication and centrifuged at 4° C. at 20,000 rpm for 20 minutes. Fusion protein was collected from the bacterial supernatant and the resulting supernatant containing the desired Mdm2 fusion protein was purified using a His-Bind® column affinity purification kit (Novagen). The resulting protein was dialyzed in 10 mM PBS with 5 mM EDTA and 0.5 mM DTT, and characterized by SDS-PAGE analysis.

Example 3

The pGEX-4T-2-p300 fusion vector was transformed into BL21(DE3) competent E.coli (Novagen) in M9 minimal media with ¹⁵NH₄Cl as the primary nitrogen source. Protein production was induced with 1 mM IPTG at OD₆₀₀ of 1 for 16 hours at 15° C. Production of the desired p300-CH1-GST fusion product was verified by SDS-PAGE. Bacteria were harvested and resuspended in the lysis buffer with 20 mM Phosphate buffer (Research Products International, Corp.), 100 μM DTT (Fisher), 100 μM ZnSO₄ (Sigma), 0.5% TritonX 100 (Sigma), 1 mg/mL Pepstatin A (Research Products International, Corp.), 10 mg/mL Leupeptin A (Research Products International, Corp.), 500 μM PMSF (Sigma), and 0.5% glycerol at pH 8.0. Pellets were lysed by sonication and centrifuged at 4° C. and 20,000 rpm for 20 minutes. Fusion protein was collected from the bacterial supernatant and purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham) prepared according to the manufacturer's directions. GST-tag was cleaved by thrombin and protein was eluted from resin. Collected fractions were assayed by SDS-PAGE gel; pooled fractions were treated with protease inhibitor cocktail (Sigma) and against a buffer containing 10 mM Tris, 50 mM NaCl, 2 mM DTT (Fisher), and 3 equivalents ZnSO₄ at pH 8.0 to ensure proper folding (vide supra).

Example 3

The pGEX-4T-2-p300 fusion vector was transformed into BL21(DE3) competent E. coli (Novagen) in M9 minimal media with ¹⁵NH₄Cl as the primary nitrogen source. Protein production was induced with 1 mM IPTG at OD₆₀₀ of 1 for 16 hours at 15° C. Production of the desired p300-CH1-GST fusion product was verified by SDS-PAGE. Bacteria were harvested and resuspended in the lysis buffer with 20 mM Phosphate buffer (Research Products International, Corp.), 100 μM DTT (Fisher), 100 μM ZnSO₄ (Sigma), 0.5% TritonX 100 (Sigma), 1 mg/mL Pepstatin A (Research Products International, Corp.), 10 mg/mL Leupeptin A (Research Products International, Corp.), 500 μM PMSF (Sigma), and 0.5% glycerol at pH 8.0. Pellets were lysed by sonication and centrifuged at 4° C. and 20,000 rpm for 20 minutes. Fusion protein was collected from the bacterial supernatant and purified by affinity chromatography using glutathione Sepharose 4B beads (Amersham) prepared according to the manufacturer's directions. GST-tag was cleaved by thrombin and protein was eluted from resin. Collected fractions were assayed by SDS-PAGE gel; pooled fractions were treated with protease inhibitor cocktail (Sigma) and against a buffer containing 10 mM Tris, 50 mM NaCl, 2 mM DTT (Fisher), and 3 equivalents ZnSO₄ at pH 8.0 to ensure proper folding (vide supra).