V51 pIRESpuro-GLUE (pGLUE)
(Plasmid #15100)

• Purpose: (Empty Backbone) tandem-affinity purification plasmid; streptavidin-(streptag) and calmodulin-(CBP) binding affinity tags
• Depositing Lab: Randall Moon
• Publication: Angers et al Nat Cell Biol. 2006 Apr . 8(4):348-57.

Backbone

• Vector backbone: pIRESpuro-GLUE
• Backbone manufacturer: Moon Lab
• Backbone size (bp): 5500
• Vector type: Mammalian Expression
• Selectable markers: Puromycin

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: None
• Tags / Fusion Proteins:
  • streptag (N terminal on backbone)
  • HA (N terminal on backbone)
  • CBP tag (N terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ sequencing primer: T7

Resource Information

• Articles Citing this Plasmid:
  • 6 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor 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.