Intracellular Staining by Flow Cytometry
|Detection of GFP in HEK293 Human Cell Line Transfected with GFP by Flow Cytometry. HEK293 human embryonic kidney cell line transfected with (A) wild-type GFP or (B) eGFP was stained with Goat Anti-GFP APC‑conjugated Antigen Affinity-purified Polyclonal Antibody (Catalog # IC4240A). Quadrant markers were set based on control antibody staining (Catalog # IC108A). To facilitate intracellular staining, cells were fixed with Flow Cytometry Fixation Buffer (Catalog # FC004) and permeabilized with Flow Cytometry Permeabilization/Wash Buffer I (Catalog # FC005). View our protocol for Staining Intracellular Molecules.|
Green Fluorescent Protein (GFP) is a 27 kDa protein originally isolated from the jellyfish Aequorea victoria. In the presence of UV light (490-520 nm), it emits a green fluorescent color that can be used to pinpoint locations of various intracellular proteins. GFP is 238 amino acids (aa) in length. It is a globular monomer that has a tendency to dimerize. The monomer has the shape of a beta -barrel with a chromophore (aa 65-67) containing alpha -helix running up its center. GFPuv is the Aequorea sequence with three aa substitutions; Phe to Ser at # 99, Met to Thr at # 153, and Val to Ala at # 163. This form expresses faster and is 18-fold brighter than native GFP; excitation peaks at 395 nm and emission at 508 nm.
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