The pGEX6P-BRD4 BD1 (aa 43-180) and BD2 (aa 342-460) constructs were generated as previously described (Vann et al., 2020 (link)). Briefly, BRD4 BD1 and BD2 constructs were expressed in E. coli BL21 (DE3) RIL in M19 minimal media supplemented with 15NH4Cl and purified as GST fusion proteins. Once the cells reached log phase, cells were induced with IPTG at a final concentration of 1 mM and grown overnight at 16 °C. The bacteria were harvested by centrifugation, resuspended in 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP and lysed by sonication. The proteins were purified from the cleared lysate using glutathione Sepharose 4B beads, and the GST tag was cleaved with PreScission or thrombin protease. The proteins were purified using a S100 column (GE Healthcare) equilibrated in 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP. The BRD4 BD1 and BD2 fractions were assessed for purity by SDS-PAGE and concentrated to ∼10-20 mg/mL.
S100 column
Manufactured by GE Healthcare
The S100 column is a size exclusion chromatography column designed for the purification of proteins and other biomolecules. It is capable of separating molecules based on their size and molecular weight. The column is made of a durable material and is suitable for use in a variety of laboratory applications.
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Lab products found in correlation
Wtc 015s5 sized exclusion column, by Wyatt Technology (1 mentions)
1260 infinity liquid chromatography system, by Agilent Technologies (1 mentions)
Dawn heleos 2 mals, by Wyatt Technology (1 mentions)
Astra 6 software, by Wyatt Technology (1 mentions)
Optilab rex differential refractive index detector, by Wyatt Technology (1 mentions)
3 protocols using s100 column
1
BRD4 BD1 and BD2 Construct Purification
2
SEC-MALS Analysis of PML RING Mutants
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The purified PML RING1–119 and mutants were subjected to gel filtration analysis (S100 column, GE Healthcare). The elution peaks, as monitored by UV absorption at 280 nm, were pooled separately and chosen for size exclusion chromatography-multi-angle light scattering ( SEC-MALS) characterization, respectively. In brief, the purified protein samples were concentrated and analyzed using a WTC-015S5 sized exclusion column (Wyatt Technology) which was connected to a 1260 infinity liquid chromatography system (Agilent Technology) equipped with inline DAWN HELEOS-II MALS and Optilab rEX differential refractive index detectors (Wyatt Technology). For each sample, a 40 μl injection volume and 0.5 ml min−1 flow rate were applied. Data were recorded and processed using ASTRA VI software (Wyatt Technology).
3
Purification of BRD4 Bromodomains
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The human pGEX6P-BRD4 BD1 (aa 43-180) and pGEX4T-BRD4 BD2 (aa 342-460) constructs were generated as previously described [28 (link)]. Briefly, BRD4 BD1 and BD2 bromodomain constructs were expressed in E. coli BL21 (DE3) RIL in M19 minimal media supplemented with 15NH4Cl and purified as GST fusions. Cells were induced with IPTG at a final concentration of 1 mM and grown overnight at 16 °C. The cells were harvested by centrifugation, resuspended in 10 mM HEPES pH 7.5, 150 mM NaCl, 1 mM TCEP, and lysed by sonication. The proteins were purified using glutathione Sepharose 4B beads, and the GST tag was cleaved with PreScission protease. The proteins were purified using a S100 column (GE Healthcare), equilibrated in 10 mM HEPES pH 7.5, 1 mM TCEP and 150 mM NaCl. The fractions of BRD4 BD1 and BD2 were assessed for purity by SDS-PAGE, buffer exchanged into PBS pH 6.8 and concentrated to ~10-20 mg/mL.
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