Intracellular Staining by Flow Cytometry
Detection of GFP in HEK293 Human Cell Line Transfected with eGFP by Flow Cytometry. HEK293 human embryonic kidney cell line transfected with eGFP was stained with either (A) Mouse Anti-GFP APC‑conjugated Monoclonal Antibody (Catalog # IC42401A) or |
(B) Mouse IgG1 Allophycocyanin Isotype Control (Catalog # IC002A). 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.