Recombinant ALK intracellular domain (ICD, amino acids 1064-1620) was expressed in Sf9 cells as a cleavable GST-tagged product using the BacPAK Baculovirus expression system (Clontech). Cells were infected at a MOI = 5 and harvested after 72 hours, as described [24 (link)] and lysed in lysis buffer (50mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM DTT, 0.5mM EDTA, 0.1% Triton-X100, and protease inhibitors: leupeptin, aprotinin, benzamidine, pepstatin A). The total lysate was loaded onto Glutathione Sepharose 4B resin (GE Healthcare) and incubated for 2 hours at 4°C with rotation. After extensive washing, the rALK-ICD was eluted by on-column overnight cleavage by GST-3C protease and concentrated using VivaSpin columns (GE Healthcare, cut-off 10kDa) to a final concentration of 0.3 mg/ml. ALK peptides were synthetized and purified by C S Bio Co., Menlo Park, CA. The full sequence of peptides is presented in Supplementary Table 1 . Each peptide was 36 amino acids long, with a 12-amino acid overlap between flanking peptides.
Bacpak baculovirus expression system
Manufactured by Takara Bio
Sourced in Japan
The BacPAK Baculovirus Expression System is a comprehensive kit developed by Takara Bio for the expression of recombinant proteins in insect cells. The system provides the necessary components, including transfer vectors, expression hosts, and reagents, to facilitate the production of target proteins. The core function of the BacPAK Baculovirus Expression System is to enable the efficient expression and purification of desired recombinant proteins in a baculovirus-insect cell expression system.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b resin, by GE Healthcare (1 mentions)
Vivaspin column, by GE Healthcare (1 mentions)
Pbacpak8 transfer vector, by Takara Bio (1 mentions)
Ni2 sepharose high performance, by GE Healthcare (1 mentions)
Quikchange site directed mutagenesis kit, by Agilent Technologies (1 mentions)
5 protocols using bacpak baculovirus expression system
1
Recombinant ALK Intracellular Domain Expression
2
Recombinant Desmoglein Ectodomains Production
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We have previously constructed and expressed the entire extracellular domains of human Dsg1 and Dsg3 using the baculovirus system (Ding et al., 1997 (link); Ding et al., 1999 (link); Flores et al., 2012 (link)). The murine Dsg3 DNA plasmid pEVmod-murineDsg3-His (kind gift of Dr. Masayuki Amagai, Keio University, Tokyo, Japan) was used to generate the mDsg3 recombinant baculovirus using the BacPAK Baculovirus Expression System (Clontech, Mountain View, CA). Soluble ectodomains of hDsg1, hDsg3, and mDsg3 were produced in the baculovirus system and purified by nickel chromatography as described (Flores et al., 2012 (link)).
3
Purification of Recombinant Flag-OGT1
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N-terminal Flag-tagged human OGT1 and NSL3 were subcloned into pBacPAK8. Recombinant baculoviruses were generated with the BacPAK baculovirus expression system (Clontech). Recombinant Flag-OGT1 from Sf21 cells were purified essentially as described [11 (link)].
4
Recombinant HA Protein Expression
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Recombinant histidine-tagged full-length HA (HA0) originated from IAV H1N1 strain A/Puerto Rico/8/1934 (PR8) was expressed in Sf21 cells using baculovirus as follows. A DNA fragment encoding full-length HA was obtained from cDNA of infected MDCK cells by PCR using specific primers (Supplementary Table. 1 ) and then cloned into a pBacPAK8 transfer vector (Clontech). A mutant construct with an amino-acid substitution of Leu194 to Ala (HA L194A) was prepared using a QuikChange site-directed mutagenesis kit (Agilent). Recombinant baculovirus was generated using the BacPAK Baculovirus Expression System (Clontech). Sf21 cells were infected with each recombinant baculovirus for 3 days and then lysed in lysis buffer (1% NP-40, 50 mm Tris-HCl pH 8.0, 135 mm NaCl, and complete protease inhibitor cocktail (Roche). The supernatants were incubated with Ni2+-sepharose high-performance (GE Life Science) for 16 h at 4 °C. After the beads were washed, each immobilized HA was prepared. For the preparation of soluble HA, immobilized HA was eluted with elution buffer (0.1% NP-40, 20 mm Tris-HCl pH 8.0, 500 mm NaCl, and 1 m imidazole) followed by desalting.
5
Recombinant Desmoglein Ectodomains Production
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