AutoFluorescent Proteins and Fusion Vectors: AFPs

AFPs^® (AutoFluorescent Proteins) are optimized variants of the Aequorea victoria green fluorescent protein. Modifications to the amino acid sequence of wtGFP have generated AFPs^® that are brighter, more efficient, and have improved excitation and emission spectra. Our SuperGlo™GFP (sgGFP™) and SuperGlo™BFP (sgBFP™) are the brightest green and blue fluorescent proteins available today.

All of our sgAFP™ vectors feature the following :

• AFPs^® designed with mutations that optimize protein folding and fluorophore formation at 37°C.
• Codon-optimized coding sequences to stabilize mRNA structure and enhance expression without restricting host range.
• 'Kozak' optimized initiation codons to encourage efficient translation.

sgGFP™: SuperGlo™ Green Fluorescent Protein

• Broadest Stoke's shift of all green variants
• Single enhanced excitation peak

sgGFP™ is the only available GFP with a single enhanced excitation peak at 474 nm. All other popular variants have excitation peaks at or near 488 nm. All of these variants fluoresce green at ~509 nm. sgGFP™ is therefore the only GFP that has a Stoke's shift that exceeds the minimum of 30 nm recommended for easy visualization and sensitive detection. sgGFP™ can be readily detected on fluorimeters, fluorescent microscopes, and by fluorescent cell sorters(FACS). sgGFP™ is also the best partner for FRET with BFP.

Mutations:

F64L - improved solubility, and protein folding at 37°C; more rapid and efficient fluorophore formation.
S65C - a single excitation peak at 474 nm.
I167T - enhanced excitation peak at 474 nm.

sgBFP™: SuperGlo™ Blue Fluorescent Protein

• Highest quantum yield of blue variants

sgBFP™ is the perfect partner for the GFP. It is brighter than other available blue variants, and its spectral properties make it easy to distinguish from GFP or YFP. sgBFP™ can be readily detected on fluorimeters, fluorescent microscopes, and by fluorescent cell sorters. Furthermore, the large overlap between the emission peak of sgBFP™ and the excitation peak of sgGFP™ make them perfect partners for FRET.

Mutations:

F64L - improved solubility, and protein folding
at 37°C; more rapid and efficient
fluorophore formation.
Y66H - a single excitation peak at 387 nm, and emission at 450 nm.
V163A - improved solubility and protein folding.

Excitation and Emission Peaks of AFPs^®

• clear images
• increased signal to noise ratio

AFP^® Fusion Vectors

Storage
-20°C

Reporter Vectors

These basic reporter vectors can be used to generate fusion proteins between the C-terminus or N-terminus of a target protein and the AFP^®.

The pQBI-pgk and pQBI-Pol II vectors can be used to generate stable transformants and transgenic animals.

sgGFP™ and sgBFP™ Cloning Vectors

In order to facilitate the use of AFPs^® as fluorescent tags, a panoply of fusion vectors are available. The pQBI25/50-fPA vectors contain short cloning sites at the N- and C-termini of the AFPs^®, to allow the cloning of exogenous coding sequences (by PCR) with minimal additions. Three series of vectors, pQBI25/50-fN 1,2,3, pQBI25/50-fA 1,2,3 and pQBI25/50-fC 1,2,3, have complete Multiple Cloning Sites (MCS) in all three open reading frames, at the N- or C-terminus of the AFPs^® coding region. These three series of vectors also have a peptide linker placed between the AFP, and the MCS to encourage independent folding of the two protein moities without hindrance. The fA series of vectors contain "Kozak" optimized initiation codons for optimal expression, whereas the fN series of vectors have no initiation codon, allowing the use of the endogenous sequences of the fusion protein. All of these vectors have been designed as functional cassettes, permitting the isolation of the CMV promoter region, AFPs^® coding sequence, the polyadenylation signal sequence, or any combination thereof. Each vector also contains the SV40 neo cassette for biochemical selection of transformed cells.

sgGFP Cloning Vectors

sgBFP Cloning Vectors

Subcellular Localization Vectors

We offer a number of sgGFP™ and sgBFP™ fusion vectors producing proteins that localize to various subcellular regions.

• Increases the sensitivity of detection
• Facilitates double labeling experiments
• Enhances the stability of the fluorescence when ethanol-based fixatives are used

AIDS Research Tools: AFP^® Fusion Proteins

• Generates fully bi-functional, localized, HIV-AFP fusion proteins
• Gag-GFP, dependent on expression of functional rev-protein in the same cell, serves as a rapid and sensitive indicator of HIV infection
• M10BLrev-AFP constructs localize to the nucleolus; M10BL defective

Anti-GFP Antibodies

We offer two monoclonal antibodies (mAb) for the detection of all of its GFPs. Anti-GFP mAbs are purified from the supernatant of mouse hybridomas. Both antibodies are highly specific for AFPs^® and recognize GFP/BFP and all other variants.

The 11E5 mAb is ideal for detection of GFP and GFP-fusion proteins in Western analyses. Use of mAb 11E5 allows detection of as little as 10 ng of GFP using colorimetric methods, and will detect picogram quantities with chemiluminescent detection assays. Performing Western blots with the 11E5 mAb allows confirmation that GFP and GFP-fusion proteins are being expressed and are of the expected molecular weight.

The 3E6 mAb is used for immunoprecipitation, immunocytochemistry, and ELISA assays. The 3E6 is an IgG2a antibody, allowing purification of GFP and GFP-fusion proteins with either protein A or protein G.

Both antibodies are highly specific for GFPs in both bacterial and mammalian cell extracts, providing clean purifications and strong clean signals.

Storage
-80°C

Recombiant Proteins

Recombinat SuperGlo™ GFP and SuperGlo™ BFP can be used as standards to calculate the level of expression and correlate it to the relative fluorescence intensity.

Storage
-20°C