EPAC1-CNB (amino acids 169–318 of EPAC1) and EPAC2-CNB (amino acids 304–453 of EPAC2) cDNAs were sub-cloned into the multi-cloning site of the pGEX-6P-1 expression vector (Invitrogen). Recombinant GST-fusion proteins were then expressed and purified from BL-21 Escherichia coli, as previously described34 (link), 38 (link). The purification of recombinant EPAC1 (149–881), EPAC2 (280–993) and Rap1b (1–167) from bacteria was done as previously described29 (link). Protein concentrations were determined by the bicinchoninic acid (BCA) assay or absorbance at 280 nm. All recombinant proteins were determined to be pure by SDS-PAGE analysis. Proteins were stored in aliquots at −80 °C until required.
Pgex 6p 1 expression vector
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The PGEX-6P-1 expression vector is a plasmid designed for the expression of recombinant proteins in Escherichia coli. It contains the tac promoter, which allows for the inducible expression of the target gene, and the glutathione S-transferase (GST) gene, which can be used for affinity purification of the expressed protein. The vector also includes multiple cloning sites, an ampicillin resistance gene, and a pBR322 origin of replication.
Automatically generated - may contain errors
2 protocols using pgex 6p 1 expression vector
1
Recombinant EPAC and Rap1b Expression and Purification
2
Recombinant Protein Purification Protocol
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Antibodies were obtained from commercial suppliers or made in house, as indicated: TPL-2 (Santa Cruz; sc-720), ABIN-2 (Lang et al., 2004 (link)), GST (Sigma; A7340), Hsp90 (Santa Cruz; sc-7947), HA (Roche; 11867423001), p-ERK1/2 (Cell Signaling; 9101), 3×FLAG (Sigma; F1804), M1 Ubiquitin (Merck; MABS451), p-p38 (Cell Signaling; 4511), ABIN-1 (MRC Protein Phosphorylation & Ubiquitylation Unit, University of Dundee, Dundee, Scotland, UK), tubulin (a gift from K. Gull; University of Oxford); NF-κB1 p105 (Cell Signaling; 4717), Actin (Santa Cruz; sc-1615), and His6 (Sigma; A7058).
cDNAs encoding human ABIN-2 WT and ABIN-2E255K proteins were sub-cloned in pGex-6P-1 expression vector (Invitrogen) by the MRC Protein Phosphorylation & Ubiquitylation Unit (University of Dundee). Recombinant proteins were expressed in Escherichia coli and purified on glutathione Sepharose 4B resin, using standard methods.
cDNAs encoding human ABIN-2 WT and ABIN-2E255K proteins were sub-cloned in pGex-6P-1 expression vector (Invitrogen) by the MRC Protein Phosphorylation & Ubiquitylation Unit (University of Dundee). Recombinant proteins were expressed in Escherichia coli and purified on glutathione Sepharose 4B resin, using standard methods.
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