AcGFP1 Fluorescent Protein

Living Colors® AcGFP1 Vectors encode a distinct green fluorescent protein that has been engineered to provide excellent performance when expressed as a fusion with your protein of interest. It is ideal for multicolor applications in flow cytometry and fluorescence microscopy.

AcGFP1 was derived from the jellyfish Aequorea coerulescens and is a novel alternative to Aequorea victoria GFP (1). AcGFP1 is an engineered, fluorescent mutant of the wild-type protein. It features an open reading frame that has been human codon optimized. This optimization increases the translational efficiency of the AcGFP1 mRNA and results in higher mammalian cell expression levels.

The AcGFP1 protein is stable, allowing you to monitor fluorescence over extended periods of time. The chromophore matures rapidly and is readily detected 8–12 hours after transfection. Like all of Clontech's fluorescent proteins, AcGFP1 can be detected in cells without adding cofactors or substrates (2–4).

The AcGFP1 protein is an ideal fluorescent tag for visualizing and tracking a protein of interest. AcGFP1 has been widely validated as a fusion tag, using a wide variety of proteins with diverse functions and subcellular locations (Figure 1). With excitation and emission spectra distinct from those of DsRed proteins (such as DsRed2 and DsRed-Monomer), AcGFP1 is particularly suited for use in multicolor applications with DsRed to simultaneously visualize the subcellular localization of two proteins of interest. It also performs well in cell-based assays that monitor protein subcellular trafficking (Figure 2), and cells are easily detected and sorted by flow cytometry (data not shown). As with all of our Living Colors Fluorescent Proteins, AcGFP1 is well tolerated by mammalian cells, and has been successfully used to establish stably transfected, clonal cell lines.

A True Monomer

The monomeric nature of the AcGFP1 protein has been confirmed by three independent methods. When recombinant AcGFP1 is analyzed by FPLC gel filtration chromatography, it elutes in a single uniform peak at a retention time consistent with a 24 kDa molecular weight (Figure 3, Panel A). Further analysis by sucrose density gradient ultracentrifugation yields a fractionation profile for AcGFP1 that is consistent with a monomeric protein (Figure 3, Panel B). Pseudonative gel electrophoresis of recombinant AcGFP1 protein in comparison to DsRed-Express (oligomeric) also supports this conclusion (Figure 3, Panel C). All of these results agree with the calculated 26.9 kDa molecular weight based on amino acid sequence.

Compatible with Existing Filter Sets and Living Colors Antibodies

AcGFP1 has an excitation maximum of 475 nm and an emission maximum of 505 nm. Its brightness and spectral properties allow AcGFP1 to be detected using existing filter sets for FITC. This includes standard factory-installed microscope filters as well as custom-tailored, optimized sets such as those available from Chroma Technology Corporation (see www.chroma.com for details). AcGFP1 can be easily detected in both Western blot and immunoprecipitation applications by our wide array of Living Colors Antibodies.

Wide Range of Vectors Available

We have a large number of AcGFP1 vectors available for multiple applications. Our collection includes source vectors and those that express AcGFP1 as either an N- or C-terminal fusion, or as a reporter (with a bicistronic internal ribosome entry sequence, IRES2). Several subcellular localization vectors— including actin and Golgi markers—are also available.