pIRES2-AcGFP1 Vector

Description

pIRES2-AcGFP1 is a bicistronic vector designed for the simultaneous expression of a novel green fluorescent protein AcGFP1 and a protein of interest from the same transcript in transfected mammalian cells. This vector contains an internal ribosome entry site IRES2 of the encephalomyocarditis virus EMCV . When a gene of interest is inserted into the MCS located upstream of the IRES, both the gene of interest and the AcGFP1 coding region will be translated from a single bicistronic mRNA. Our products are to be used forResearch Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval. Back AcGFP1 is a monomeric protein.Panel A. Recombinant AcGFP1 protein was analyzed by FPLC gel filtration chromatography. Overall protein absorbance A280 and chromophore excitation A477 of the eluted material were monitored simultaneously. AcGFP1 elutes from the column at a retention time corresponding to a molecular weight of 24 kDa. The calculated molecular weight of AcGFP1 is 26.9 kDa.Panel B. Recombinant AcGFP1 protein was analyzed by sucrose density ultracentrifugation using a continuous gradient.Panel C. Pseudonative gel analysis of proteins. The oligomeric structure of proteins is preserved during SDS-PAGE analysis if samples are kept at 4C and not boiled prior to loading on a gel. Boiled and unboiled recombinant proteins 7.5 mug were separated by SDS-PAGE electrophoresis 12 % acrylamide . In both the boiled denatured and unboiled nondenatured samples, AcGFP1 runs as a uniform band of 30 kDa due to its monomeric structure. The unboiled nondenatured DsRed-Express runs at a much higher molecular weight than its denatured boiled counterpart due to its oligomeric structure. Back Use of AcGFP1 for fusions and fluorescence microscopy applications.Panels AandB. Activation of Protein Kinase C alpha was monitored with Living Colors AcGFP1.Panel A. HEK 293 cells were stably transfected with a plasmid encoding AcGFP1 fused to PKC alpha.Panel B. Cells were induced with 1.5 mug/ml PMA for 3 min. The PKC alpha-AcGFP1 fusion moves from the cytosol to the plasma membrane, a result consistent with the known mobilization pattern of PKC alpha.Panel C. HeLa cells were transiently transfected with pAcGFP1-Actin and visualized by fluorescence microscopy. Back Back Back