GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Information and tips on how to use GFP-tagged proteins in cell biology - [Read How to Use GFP-Tagged Proteins in Cell Biology]

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Protocol measuring the amount of GFP expression and DNA content in permeabilized cells. Includes: Fix cells with formaldehyde; Permeabilize cells with ethanol; Preparation of Buffered 2% Formaldehyde Solution. - [Read Measurement of GFP Expression and DNA Content in Permeabilized Cells]

Green fluorescent protein is commonly used to monitor gene expression and protein trafficking within intact cells. The Monster Green® Fluorescent Protein is encoded by an improved synthetic version of the green fluorescent protein gene originally cloned from Montastrea cavernosa (Great Star Coral). - [Read Monster Green® Fluorescent Protein Assay]

Protocol specifically describes data acquisition for a particular variant of GFP (EGFP) or Oregon Green as a donor fluorophore, but it can be adapted for image acquisition of other chromophore. - [Read Probing Protein Interactions Using GFP and FRET]

In preparation for FLIM-FRET analysis, the appropriate donor and acceptor components must be introduced into live or fixed cells. The method of introduction depends on the nature of the components and the state of the cells. For example, plasmid DNAs encoding a protein of interest fused to a variant of GFP may be introduced into live cells by transfection or microinjection, whereas labeled antibodies are delivered by microinjection. - [Read Probing Protein Interactions Using GFP and FRET Protocol]

Describes how components (usually Fab fragments of antibodies) that are to be introduced into cells are labeled with fluorescent sulfoindocyanine dyes. - [Read Probing Protein Interactions Using GFP and FRET.]