V51 pIRESpuro-GLUE (pGLUE)
Purpose(Empty Backbone) tandem-affinity purification plasmid; streptavidin-(streptag) and calmodulin-(CBP) binding affinity tags
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||15100||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerMoon Lab
- Backbone size (bp) 5500
Vector typeMammalian Expression
Growth in Bacteria
Copy numberHigh Copy
/ Fusion Proteins
- streptag (N terminal on backbone)
- HA (N terminal on backbone)
- CBP tag (N terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ sequencing primer T7 (Common Sequencing Primers)
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.
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.
For your Materials & Methods section:V51 pIRESpuro-GLUE (pGLUE) was a gift from Randall Moon (Addgene plasmid # 15100 ; http://n2t.net/addgene:15100 ; RRID:Addgene_15100)
For your References section:The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation. Angers S, Thorpe CJ, Biechele TL, Goldenberg SJ, Zheng N, MacCoss MJ, Moon RT. Nat Cell Biol. 2006 Apr . 8(4):348-57. 10.1038/ncb1381 PubMed 16547521