Purification of DDB1ΔBPB

Purification of full length DDB1 and DDB1ΔBPB

1. Human full length DDB1 and DDB1ΔBPB (removing residues 400-704) was cloned into pFastBac with an N-terminal Strep-tag and an 8×His tag.
2. Baculovirus of human full length DDB1 and DDB1ΔBPB was produced following the Bac-to-Bac^TM baculovirus expression system user guide (1).
3. Sf9 cells was infected with the baculovirus (optimal baculovirus volume and infection time determination were needed).
4. After baculovirus infection, Sf9 cells were lysed by sonication in binding buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% glycerol, 2 mM TCEP, 1 mM PMSF, 10 mM imidazole) supplemented with 1x cOmplete, Mini, EDTA-free protease inhibitor cocktail (Roche).
5. Cell lysates were clarified by 20000g centrifugation for 1hr at 4°C;
6. DDB1 full length and DDB1ΔBPB protein was purified from clarified lysates by Ni NTA Beads and eluted in 50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10% glycerol, 2 mM TCEP, 1 mM PMSF, 300 mM imidazole.
7. The fractions containing DDB1ΔBPB were pooled, concentrated, and then subjected to gel filtration on an ENrich SEC650 size exclusion column (Bio-Rad) in 50 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM TCEP.

Reference

(1)https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0000414_BactoBacExpressionSystem_UG.pdf&title=VXNlciBHdWlkZTogQmFjLXRvLUJhYyBCYWN1bG92aXJ1cyBFeHByZXNzaW9uIFN5c3RlbQ==

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