Paul.xu: Created page with "Expression and purification of His-tagged Xis and its derivatives. E. coli
strains DH5␣, harboring derivatives of the pYL-XisHR plasmid, and
BL21(DE3), with the pYL-XisHP..."

2012-04-02T01:30:47Z

Created page with "Expression and purification of His-tagged Xis and its derivatives. E. coli strains DH5␣, harboring derivatives of the pYL-XisHR plasmid, and BL21(DE3), with the pYL-XisHP..."

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Expression and purification of His-tagged Xis and its derivatives. E. coli strains DH5␣, harboring derivatives of the pYL-XisHR plasmid, and BL21(DE3), with the pYL-XisHP plasmid, were used for expression of the Xis mutants and the wild-type Xis protein, respectively. Cells were grown to mid-log phase in Luria-Bertani medium containing the appropriate antibiotics. Produc- tion of His-tagged proteins was induced by adding IPTG to a final concentration of 1 mM, followed by growth for 3 h at 37°C. After centrifugation, harvested cells were disrupted by treatment in a French pressure cell in 50 mM sodium phos- phate buffer, pH 7.4, with 300 mM NaCl. Prior to disruption, 250 ␮l of protease inhibitor cocktail for bacterial cell extracts (Sigma product no. P8465) was added per gram of cell mass. Cell lysates were centrifuged at 12,000 ϫ g for 30 min, and the supernatants were mixed with nickel-nitrilotriacetic acid column material (Qiagen) saturated in 50 mM sodium phosphate buffer, pH 7.4, with 300 mM NaCl and 10% glycerol. The column was washed extensively, and the His-tagged proteins were eluted by adding 250 mM imidazole in the same buffer. The total protein concentrations were measured using the dye-binding assay (3). The amount of Xis in each protein sample was determined by measuring peaks after scanning the Coomassie blue-stained sodium dodecyl sulfate-polyacrylamide gel. 

[[Category:Protocols]]

Excisionase Purification - Revision history

Paul.xu: Created page with "Expression and purification of His-tagged Xis and its derivatives. E. colistrains DH5␣, harboring derivatives of the pYL-XisHR plasmid, andBL21(DE3), with the pYL-XisHP..."

Paul.xu: Created page with "Expression and purification of His-tagged Xis and its derivatives. E. coli
strains DH5␣, harboring derivatives of the pYL-XisHR plasmid, and
BL21(DE3), with the pYL-XisHP..."