Addgene - V51 pIRESpuro-GLUE (pGLUE) Plasmid Data

Plasmid 15100: V51 pIRESpuro-GLUE (pGLUE)

Gene/insert name:
None
Fusion protein or tag:
streptag
Terminal:
N terminal on backbone
Fusion protein or tag:
HA
Terminal:
N terminal on backbone
Fusion protein or tag:
CBP tag
Terminal:
N terminal on backbone
Vector backbone:
pIRESpuro-GLUE
(Search Vector Database)
Backbone manufacturer:
Moon Lab
Vector type:
Mammalian Expression
Backbone size (bp):
5500
5' sequencing primer:
T7 List of Sequencing Primers
Bacterial resistance(s)
Ampicillin
Growth strain(s)
DH5alpha
Growth temperature (℃):
37
High or low copy:
High Copy
Selectable markers:
Puromycin
Sequence: View sequences (2)
Map:
View map
Map:
View map
Principal Investigator:
Randall Moon
Terms and Licenses:
MTA

Comments:

To generate the pGLUE tandem-affinity purification plasmid, the pIRES–puro (Clontech, Mountain View, CA) was used as the backbone vector and the dual affinity tag was constructed using PCR.

The pGLUE expression vector was designed to have the streptavidin-(streptag) and calmodulin-(CBP) binding affinity tags placed in tandem at the N-terminus of the bait protein. An HA epitope is also present to facilitate protein detection. The first round of affinity purification is performed using a streptavidin resin, followed by the specific elution with biotin and the protease TEV. The second round is done using the calcium dependent binding to the calmodulin resin and specific elution by chelation using EGTA.

Addgene has sequenced a portion of this plasmid for verification. Click here for the sequencing result.

Article: The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Angers et al (Nat Cell Biol. 2006 Apr . 8(4):348-57. PubMed)

Please acknowledge the principal investigator and cite this article if you use this plasmid in a publication. Also, please include the text "Addgene plasmid 15100" in your Materials and Methods section.