The pCMV-HA Mammalian Expression Vector expresses proteins containing an N-terminal hemagglutinin (HA) epitope tag. The HA epitope tag is well-characterized and highly immunoreactive. High-level expression in mammalian cells is driven from the human cytomegalovirus immediate early promoter/enhancer (PCMV IE). The vector contains an intron (SV40 splice donor/splice acceptor); the epitope tag; an MCS; and a polyadenylation signal from SV40. This vectors also possesses the ampicillin resistance gene for selection in E. coli.

载体用途

To create a fusion of a gene of interest and the HA tag, insert the gene into the MCS in frame with the HA coding sequence. The resulting HA-tagged proteins can be identified with the HA-Tag Polyclonal Antibody (IgG), provided with this vector, or another antibody raised against the HA tag. The epitope tag is also useful for facilitating purification of the protein, identifying associated proteins, characterizing new proteins by immunoprecipitation, and determining subcellular localization. The MCS in this vector is compatible with the MCSs in Clontech's Matchmaker™ Two-Hybrid System Vectors. Compatibility with System 3 Vectors is noted in the MCS diagram. Consult the Vector Information Packet provided with any Matchmaker vector for complete information.

After obtaining putative positive clones in your Matchmaker two-hybrid screen, use the pCMV-Myc and pCMV-HA Vectors to verify the interactions identified in yeast directly in mammalian cells. To accomplish this, subclone the selected inserts into the pCMV-Myc Vector and the "bait" insert into the pCMV-HA Vector. Alternatively, clone the "bait" insert into pCMV-Myc and the library inserts into pCMV-HA. To confirm predicted interactions in vivo via coimmunoprecipitation, cotransfect pCMV-Myc with the pCMV-HA Vector into mammalian cells and immunoprecipitate using the c-Myc Monoclonal or HA-Tag Polyclonal Antibody provided with the vectors.