An Ig κ-chain signal sequence (METDTLLLWVLLLWVPGSTGDG), AP tag (GGLNDIFEAQKIEWHEGATG) and SEP were cloned in-frame with the 5'-end of the coding sequence for the mature rat GluA1 and 2 subunit proteins. The entire open reading frames (ORFs) were cloned upstream (5’) of an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence. The BirA-ER coding sequence30 (link) (a gift from A. Ting, MIT Cambridge) was then cloned to the 3' end of the IRES such that the start codon of the BirA-ER signal sequence corresponded to the 11th ATG of the IRES sequence. Doxycycline-dependent expression of the resulting dual-construct bAP::SEP::GluA was achieved by cloning the entire AP::SEP::GluA IRES BirA-ER sequence into the multiple cloning site of the pBI vector (BD Bioscience) and co-transfecting it with approximately equal molar quantities of rtTA-transactivator. The GluA2 subunit used was edited at the Q/R site (R607), unedited at the R/G site (R764) and the ligand-binding domain splice variant was flop except for residue S775 (flip), where amino acid numbering corresponds to the coding sequence of the immature GluA2 peptide (NP_001077280.1). The GluA1 subunit used was flip splice variant. Plasmid DNA was prepared using endotoxin-free MaxiPreps (Qiagen). All constructs were verified by restriction enzyme digest patterns and Sanger DNA sequencing.
Pbi vector
Manufactured by BD
The PBI vector is a laboratory instrument used for the purification of nucleic acids, such as DNA and RNA, from a variety of biological samples. It utilizes a proprietary polymer-based technology to capture and isolate the target molecules, enabling their subsequent analysis or downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using pbi vector
1
Engineered GluA1/2 Subunit Expression
2
Engineered GluA1/2 Subunit Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
An Ig κ-chain signal sequence (METDTLLLWVLLLWVPGSTGDG), AP tag (GGLNDIFEAQKIEWHEGATG) and SEP were cloned in-frame with the 5'-end of the coding sequence for the mature rat GluA1 and 2 subunit proteins. The entire open reading frames (ORFs) were cloned upstream (5’) of an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) sequence. The BirA-ER coding sequence30 (link) (a gift from A. Ting, MIT Cambridge) was then cloned to the 3' end of the IRES such that the start codon of the BirA-ER signal sequence corresponded to the 11th ATG of the IRES sequence. Doxycycline-dependent expression of the resulting dual-construct bAP::SEP::GluA was achieved by cloning the entire AP::SEP::GluA IRES BirA-ER sequence into the multiple cloning site of the pBI vector (BD Bioscience) and co-transfecting it with approximately equal molar quantities of rtTA-transactivator. The GluA2 subunit used was edited at the Q/R site (R607), unedited at the R/G site (R764) and the ligand-binding domain splice variant was flop except for residue S775 (flip), where amino acid numbering corresponds to the coding sequence of the immature GluA2 peptide (NP_001077280.1). The GluA1 subunit used was flip splice variant. Plasmid DNA was prepared using endotoxin-free MaxiPreps (Qiagen). All constructs were verified by restriction enzyme digest patterns and Sanger DNA sequencing.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!