The bacteria expression plasmids pET-28a containing cDNAs of different Sox2 deletions, Ddx5, and RNaseH1 fusing to N-terminal tagged 6× Histidine were expressed into transetta (DE3) chemically competent cells (Transgene Biotech, CD801). Transformed cells were grown at 37°C to a density of 0.6 to 0.8 at OD600 (optical density at 600 nm) and induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside at 25°C for 6 hours. The cells were collected and resuspended in lysis buffer [25 mM tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, and 20 mM imidazole]. The cells were lysed and protein supernatants were allowed to flow through a Ni-NTA Sefinose Column (Sangon Biotech, C600791). The columns were washed with lysis buffer and 100 mM imidazole. The proteins were eluted with elution buffer [25 mM tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, and 300 mM imidazole]. To remove imidazole, the eluted fraction was dialyzed in dialysis buffer [25 mM tris-HCl (pH 7.5), 150 mM NaCl, and 10% glycerol] and stored at −80°C.
Cd801
Manufactured by Transgene
The CD801 is a laboratory instrument designed for cell culture and analysis applications. It provides automated cell counting and viability assessment using trypan blue dye exclusion. The CD801 can accurately measure cell density and viability in a variety of cell types.
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Lab products found in correlation
3 protocols using cd801
1
Purification of Recombinant Histidine-Tagged Proteins
2
Recombinant Protein Expression and Purification
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Protein recombination and purification were performed as described previously22 (link),38 (link). Briefly, recombinant proteins were expressed in E. coli strain BL21 [Transetta (DE3) chemically competent cell (Transgen biotech, CD801)]. In brief, 5 μL Luria-Bertani (LB) culture supplemented with 100 μg/μL ampicillin was inoculated with a single colony at 37 °C. After overnight growth, the culture was diluted 100-fold into 300 mL LB supplemented with 100 μg/mL ampicillin. Protein expression was induced in the presence of 0.4 mM IPTG at 16 °C overnight. Then the cell pellets were collected by centrifugation at 5000 rpm, 4 °C for 10 min and purified recombinant proteins using a His or Flag tag Protein Purification Kit (BeaverBeads™) according to the manufacturer’s instruction.
3
Recombinant Protein Expression in E. coli
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Recombinant proteins were expressed in E. coli strain BL21 [Transetta (DE3) chemically competent cell (Transgen biotech, CD801)]. In brief, 5 mL Luria-Bertani (LB) culture supplemented with 100 μg/mL ampicillin was inoculated with a single colony at 37 °C. After overnight growth, the culture was diluted 100-fold into 300 mL LB supplemented with 100 μg/mL ampicillin. Protein expression was induced in the presence of 0.4 mM IPTG at 16 °C overnight. Then the cell pellets were collected by centrifugation at 5000 rpm, 4 °C for 10 min and purified recombinant proteins using a His or GST tag Protein Purification Kit (BeaverBeads™) according to the manufacturer’s instruction.
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