CloneID: cAbGFP4
Antigen Long Description: The original antibody was generated by immunizing alpaca with GFP. A phage display library was constructed and the antibody was selected by panning against GFP.
Buffer Composition: PBS only.
Uniprot Accession No.: P42212
Specificity Statement: The antibody is specific for green fluorescent protein. The antibody binds wtGFP in a frontwise manner at an exposed loop region between GFP β-strands 6 and 7 as well as parts of β-strand 8.
Application Notes (Clone): The antibody could enhance GFP fluorescenceincrease by a factor of 10 in vitro. A comparable fluorescence modulation was also observed after addition of the antibody to soluble cell extract derived from human embryonic kidney (HEK) 293T cells expressing eGFP. The crystal structure of the GFP–antibody complex was determined. The dissociation constant of the antibody was measured (Kd= 0.59 nM). The fluorescence-enhancement effect induced by binding of GFP to the antibody was used to track inducible translocation of the human estrogen receptor in HeLa-Kyoto cell line (Kirchhofer et al., 2010; PMID: 20010839). In order to improve the affinity for GFP two antibodies that recognize different portions of GFP were linked together. In particular, the antibody was fused together with LaG16 through a (GGGGS)4 linker. The binding affinity of LaG16, GFP-enhancer and the construct with the (GGGGS)4 linker to GFP was measured by ITC (Kd= 6.7, 24.3 and 0.5 nM respectively).Further, the GFP-enhancer-(GGGGS)4-LaG16 chimeric nanobody was covalently linked to NHSactivated agarose and used for protein purification. The purification of the membrane protein GFP-zfP2X4 showed that the chimeric nanobody performed better than the single the antibody Enhancer alone (Zhang et al., 2020). The antibody detected protein extracts of GFP from 293T cells by western blot analysis. The antibody detected GFP in human 293T cells (US20130323747). The binding affinity of the VHH fragment to GFP was measured by surface plasmon resonance (Kd= 0.32 nM) (Saerens et al., 2005; PMID: 16095608). The VHH fragment was coupled microbubbles and the ability of μB-cAbGFP4 to recognize eGFP was confirmed by fluorescence microscopy (Hernot et al. 2012; PMID: 22197777).
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