The protein domain microarray was conducted by the Protein Array and Analysis Core (PAAC) at MD Anderson Cancer Center. A comprehensive library of human protein domains that potentially read methyllysine marks was cloned into a pGEX vector by Biomatik (Cambridge, Canada) using gene synthesis to best optimize the open reading frames for bacterial expression. Escherichia coli was used to express the protein domains as GST fusions, which were purified using glutathione-Sepharose beads. The recombinant domains were arrayed onto nitrocellulose-coated glass slides (Oncyte®Avid slides, Grace Bio-Labs, Bend, OR), using an Aushon 2470 pin microarrayer, as previously described (Espejo et al., 2002 (link)). Biotinylated compounds were pre- conjugated to streptavidin-Cy3 to generate fluorescent probes. Following probing, fluorescent signal was detected with a GenePix 4200A Microarray Scanner (Molecular Devices).
Pgex vector
Manufactured by Biomatik
Sourced in Canada
The PGEX vector is a plasmid commonly used for the expression of recombinant proteins in Escherichia coli. It contains the tac promoter, which allows for the inducible expression of the target gene. The vector also includes a glutathione S-transferase (GST) tag, which can be used for the purification of the expressed protein.
Automatically generated - may contain errors
2 protocols using pgex vector
1
Protein Domain Microarray Analysis
2
Protein Domain Microarray Analysis
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The protein domain microarray was conducted by the Protein Array and Analysis Core (PAAC) at MD Anderson Cancer Center. A comprehensive library of human protein domains that potentially read methyllysine marks was cloned into a pGEX vector by Biomatik (Cambridge, Canada) using gene synthesis to best optimize the open reading frames for bacterial expression. Escherichia coli was used to express the protein domains as GST fusions, which were purified using glutathione-Sepharose beads. The recombinant domains were arrayed onto nitrocellulose-coated glass slides (Oncyte®Avid slides, Grace Bio-Labs, Bend, OR), using an Aushon 2470 pin microarrayer, as previously described (Espejo et al., 2002 (link)). Biotinylated compounds were pre- conjugated to streptavidin-Cy3 to generate fluorescent probes. Following probing, fluorescent signal was detected with a GenePix 4200A Microarray Scanner (Molecular Devices).
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