NT-GFP Fusion TOPO™ Expression Kit

pBlue-TOPO contains a cryptic prokaryotic promoter upstream of the lacZ reporter gene, due to which E. coli transformants may appear to be light blue when screened on plates containing X-Gal. Hence, we do not recommend using pBlue-TOPO to evaluate promoter function in E. coli. However, pGlow-TOPO can be used for these studies. Note that background expression of beta-galactosidase from pBlue-TOPO does not occur in mammalian cells.

Yes, we do offer the pBlue-TOPO and pGlow-TOPO vectors that facilitate cloning of the DNA sequence of interest directly upstream of either the b-galactosidase or Cycle 3 GFP gene, respectively.
pBlue-TOPO is ideal for functional analysis of promoters with low transcriptional activity, since assays for beta-galactosidase are easy to perform and are quantitative at very low levels of expression. pGlow-TOPO is ideal for non-invasive analysis of promoter elements within intact, living cells. The fluorescent property of Cycle 3 GFP allows in vivo detection in virtually any cell type or species using microscopy with wild-type GFP filter sets or by fluorescence-activated cell sorting methods.

We recommend looking for GFP fluorescence before staining for beta-galactosidase. This is because the beta-galactosidase staining process produces a very high autofluorescence that will interfere with detection of GFP fluorescence.

Cycle 3 GFP fluorescence can be detected using a filter set designed to detect wild-type GFP (since they have the same fluorescence spectra). In-house, we use the XF76 filter set from Omega Optical. For Cycle 3 GFP, excite at 395 nm and read emission at 507 nm. You can also look at the emission spectra and record emissions from 200-800 nm.

Cycle 3 GFP fluorescence can be quantitated with any type of fluorometer with the appropriate filters and cut-off wavelengths. In-house, we have a Hitachi F-2000 Fluorescence Spectrophotometer. Our general protocol using this machine is as follows:
Dilute samples in PBS (although Tris or water would be okay). The amount of lysate to be used will of course depend upon the concentration of GFP. This will have to be determined empirically. The primary consideration is that one needs to be in the linear range of the fluorometer. We have used quantities from 5-50 µL in 1 mL of PBS in a cuvette. If readings are going to be internally compared, the most consistent results will be obtained if the amounts of lysate used are normalized to either the transfection efficiency or the total protein concentration.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

EmGFP, YFP, CFP, and BFP can be detected using standard FITC filter sets and settings. However, for optimal detection of the fluorescence signal, filter sets optimized for detection within the excitation and emission ranges for each fluorescent protein are recommended. The recommended filter sets are as follows: EmGFP: Omega filter set XF100 YFP: Omega filter set XF1042 Chroma filter set 41028 CFP: Omega filter set XF114 Chroma filter set 31044 BFP: Omega filter set XF10 Chroma filter set 31021 For information on obtaining filter sets, please contact Omega Optical, Inc. (www.omegafilters.com) or Chroma Technology Corporation (www.chroma.com) directly.