These Thermo Scientific pCMV Vectors include epitope-tagged (HA or c-Myc) and Tluc-tagged (TurboLuc Luciferase) multiple cloning site mammalian expression vectors for use in Pierce quantitative IP (qIP) assays.
Pierce qIP Assays are designed to measure the interaction of two proteins (X and Y) that are transiently co-expressed in mammalian cells as epitope-tagged and luciferase-tagged fusions, respectively. The specific luciferase required is Thermo Scientific TurboLuc (Tluc) Luciferase, which is especially small and bright. Protein interactions are quantified by measuring Tluc Luciferase activity following immunoprecipitation (IP) of epitope-tagged proteins with anti-epitope agarose or magnetic beads. As such, the assay system requires that the genes for proteins-of-interest X and Y be cloned as fusions with epitope (e.g., HA or c-Myc) and Tluc tags, respectively. Several vectors, all based on a common pCMV-MCS map, are available for this purpose. These optimized MCS vectors include varieties with tags at the N- or C-terminus. Also available are ready-to-use, positive and negative control vectors containing genes for BAD, RFP or Bcl-xL proteins.
CMV (Cytomegalovirus) promoter for constitutive expression of proteins in mammalian cells
Shared multiple cloning site (MCS) in all vectors provides convenience and versatility for transfer of cDNA gene from one plasmid to another
Tluc tagged vectors use the small and bright TurboLuc Luciferase gene, providing high expression in mammalian systems and the sensitivity required for quantitative IP
HA epitope tag (YPYDVPDYA) and Myc epitope tag (EQKLISEEDL) varieties available
N-terminal and C-terminal tagged vectors offered for each tag, enabling optimization of protein function for protein:protein interaction
Kanamycin/neomycin marker for drug selection in both bacterial and mammalian cells
Complete cloning and subcloning guide available (Tech Tip #77)
All Pierce qIP Vectors contain the same multiple cloning site (MCS) sequences. Therefore, once a gene of interest is successfully cloned into one qIP vector with the proper reading frame, the gene can be moved to another qIP vector without additional PCR amplification. This feature provides the convenient and flexible option to subclone a gene from an N-terminal tag vector to C-terminal tag vector.
BAD and Bcl-xL proteins interact strongly as a protein interaction pair. Thus, pairing an epitope-tagged BAD vector with a Tluc-tagged Bcl-xL vector forms a positive control for Pierce qIP Assays. Red fluorescent protein (RFP) does not interact with either BAD or Bcl-xL. Therefore, pairing an epitope-tagged RFP vector with Tluc-tagged Bcl-xL vector provides a negative control for Pierce qIP Assays. The prepared epitope-tagged control vectors (BAD, RFP) that are included in the Pierce qIP Kits are cloned into the nominal MCS expression vectors using NotI and BglII restriction sites. As such, these control vectors can be used as expression vectors for cloning or subcloning a gene of interest.