Pdisplay bira er
The PDisplay-BirA-ER is a plasmid that can be used to biotinylate proteins within the endoplasmic reticulum of cells. The plasmid encodes the BirA enzyme, which catalyzes the attachment of biotin to proteins containing a specific peptide sequence.
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6 protocols using pdisplay bira er
Engineered Connexin43 Constructs for Cellular Analysis
Transfection and Purification of MET Constructs
In separate experiments, pHLsec-METΔ7–8ED-BAPHIS or pHLsec-METED-BAPHIS were cotransfected in a 1:1 ratio with the biotin-ligase expression construct pDISPLAY-BirA-ER (Addgene) in HEK293T cells and cells were cultured in the presence of 10 μM biotin (Sigma-Aldrich, St Louis, MO, USA). After 48 h, cytosolic extracts were made in RIPA buffer, and medium (1 ml) was mixed with 100 μl Ni–NTA Sepharose slurry (IBA, Goettingen, Germany). After a 1 h incubation at 4 °C, Ni-beads were washed with buffer (500 mM NaCl, 50 mM phosphate buffer, pH 7.4) and loaded onto a poly-prep column (Bio-Rad, Hercules, CA, USA). After washing off specifically bound proteins with 2 ml 10 mM imidazole, His-tagged an biotinylated MET extracellular domains were eluted with 0.5 ml 0.5 M imidazole, and dialysed o/n at 4 °C to 50 mM TRIS/150 mM NaCl pH 7.5.
AviTag-GluR2/eGFP/BirA Plasmid Construction
Recombinant SARS-CoV-2 RBD Production
Immobilization and Biotinylation of Proteins
Recombinant SARS-CoV-2 RBD Expression
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