E. coli ClpP, single-chain E. coli ClpX variants, and ssrA-tagged GFP substrates were expressed as described22 (link),26 (link),33 (link). Briefly, proteins were purified by Ni+2-NTA (Qiagen) affinity chromatography, desalted into a low ionic strength buffer on a PD-10 column (GE Healthcare), purified further by ion-exchange chromatography, run on a HiLoad 16/60 Superdex 200 size-exclusion column (Amersham), and stored frozen at −80 °C in PD buffer (25 mM HEPES, pH 7.6, 100 mM KCl, 20 mM MgCl2, 10% glycerol (v/v)). Degradation assays were performed at 30 °C in PD buffer supplemented with 4 mM ATP and a regeneration system consisting of 16 mM creatine phosphate (MP Biomedicals) and 0.32 mg/mL creatine phosphokinase (Sigma). GFP degradation and extraction of the ssrA-tagged strand of split SFGFP-10/11-ssrA were quantified by loss of 511-nm fluorescence after excitation at 400 or 467 nm26 (link). Rates of ATP hydrolysis were determined using an NADH coupled assay in PD buffer at 30 °C34 (link). The binding of ClpX variants to ClpP was assayed by changes in the rate of cleavage of a decapeptide35 (link).
Hiload 16 60 superdex 200 size exclusion column
Manufactured by Cytiva
The HiLoad 16/60 Superdex 200 size-exclusion column is a laboratory equipment used for the separation and purification of proteins and other macromolecules based on their size and molecular weight. It is designed to provide high-resolution chromatographic separations with a separation range of 10,000 to 600,000 daltons.
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Lab products found in correlation
Pd 10 column, by GE Healthcare (2 mentions)
Creatine phosphate, by MP Biomedicals (2 mentions)
Creatine phosphokinase, by Merck Group (2 mentions)
Ni2 nta, by Qiagen (2 mentions)
Protease inhibitor cocktail, by New England Biolabs (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Bl21 de3 competent cells, by New England Biolabs (1 mentions)
3 protocols using hiload 16 60 superdex 200 size exclusion column
1
Purification and Characterization of E. coli ClpP and ClpX
2
Purification and Characterization of E. coli ClpP and ClpX
3
Recombinant Mouse NPM2 Purification
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Mouse NPM2 expression was carried out in BL21 (DE3) competent cells (NEB, Ipswich, MA).
After induction with 1 mM IPTG, cells were grown for 3 h at 37 °C. The culture was then centrifuged at 8000 x g for 10 min at 4 °C, and the pellets were re-suspended in one-tenth the volume of 20 mM Tris–HCl (pH 8.5), 500 mM NaCl, 1 mM EDTA containing 1:100 protease inhibitor cocktail (Roche, Indianapolis, IN). The homogenate was lysed with a French Press (SIM Aminco, Rochester, NY) and centrifuged at 8000×g for 10 min. The supernatant was then passed through a 0.45 μm filter, supplemented with 1:50 protease inhibitor cocktail, and loaded onto the IMPACT® Intein chitin resin column (NEB, Ipswich, MA) at 4 °C with a flow rate of 0.5 mL/min, and eluted under the conditions described by the supplier. Fractions containing mouse NPM2 were pooled and dialyzed for 4 h against 50 mM Tris–HCl (pH 8), 100 mM NaCl buffer at 4 °C. The dialysate was concentrated and FPLC fractionated using the ATKA® FPLC System (Amersham Pharmacia Biotech, Piscataway, NJ), using a HiLoad 16/60 Superdex® 200 Size Exclusion Column. Full length Xenopus laevis NPM2 was purified as described previously [12] (link).
After induction with 1 mM IPTG, cells were grown for 3 h at 37 °C. The culture was then centrifuged at 8000 x g for 10 min at 4 °C, and the pellets were re-suspended in one-tenth the volume of 20 mM Tris–HCl (pH 8.5), 500 mM NaCl, 1 mM EDTA containing 1:100 protease inhibitor cocktail (Roche, Indianapolis, IN). The homogenate was lysed with a French Press (SIM Aminco, Rochester, NY) and centrifuged at 8000×g for 10 min. The supernatant was then passed through a 0.45 μm filter, supplemented with 1:50 protease inhibitor cocktail, and loaded onto the IMPACT® Intein chitin resin column (NEB, Ipswich, MA) at 4 °C with a flow rate of 0.5 mL/min, and eluted under the conditions described by the supplier. Fractions containing mouse NPM2 were pooled and dialyzed for 4 h against 50 mM Tris–HCl (pH 8), 100 mM NaCl buffer at 4 °C. The dialysate was concentrated and FPLC fractionated using the ATKA® FPLC System (Amersham Pharmacia Biotech, Piscataway, NJ), using a HiLoad 16/60 Superdex® 200 Size Exclusion Column. Full length Xenopus laevis NPM2 was purified as described previously [12] (link).
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