The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration. PubMed: 20824308
1/25000. Detects a band of approximately 27 kDa (predicted molecular weight: 27 kDa). An unconjugated version of this antibody is available as ab6556.
FITC conjugated version: ab66180.
HRP conjugated version: ab69312.
Sepharose version: ab69314.
Magnetic beads version: ab69315.
Function: Energy-transfer acceptor. Its role is to transduce the blue chemiluminescence of the protein aequorin into green fluorescent light by energy transfer. Fluoresces in vivo upon receiving energy from the Ca2+ -activated photoprotein aequorin.
Subunit structure: Monomer.
Tissue specificity: Photocytes.
Post-translational modification: Contains a chromophore consisting of modified amino acid residues. The chromophore is formed by autocatalytic backbone condensation between Ser-65 and Gly-67, and oxidation of Tyr-66 to didehydrotyrosine. Maturation of the chromophore requires nothing other than molecular oxygen.
Biotechnological use: Green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors. Fluorescent proteins and its mutated allelic forms, blue, cyan and yellow have become a useful and ubiquitous tool for making chimeric proteins, where they function as a fluorescent protein tag. Typically they tolerate N- and C-terminal fusion to a broad variety of proteins. They have been expressed in most known cell types and are used as a noninvasive fluorescent marker in living cells and organisms. They enable a wide range of applications where they have functioned as a cell lineage tracer, reporter of gene expression, or as a measure of protein-protein interactions. Can also be used as a molecular thermometer, allowing accurate temperature measurements in fluids. The measurement process relies on the detection of the blinking of GFP using fluorescence correlation spectroscopy.
Sequence similarities: Belongs to the GFP family.
Biophysicochemical properties: Absorption: Abs(max)=395 nm
Exhibits a smaller absorbance peak at 470 nm. The fluorescence emission spectrum peaks at 509 nm with a shoulder at 540 nm.
Green fluorescent protein antibody
Western blot - GFP antibody (Biotin) (ab69313)
Predicted band size: 27 kDa Observed band size: 27 kDa
Western blots results from a 12.5% SDS-PAGE gels loaded with highly purified GFP (antigen) or transiently transfected COS-7 cell lysates expressing truncated Yes-GFP (1) or empty vector as shown. Membranes were probed with rabbit anti-GFP-biotin conjugate at a ratio of 1/25,000 (100 ng/2.5 ml), followed by NeutrAvidin™-HRP conjugate at a ratio of 1/50,000 (100 ng/5 ml) and ECL. Developed membranes were exposed to Kodak X-OMAT™ LS film for 15 seconds. Shown on the right is the PVDF membrane stained with Coomassie blue.
Lanes 1-3: PVDF membrane probed with rabbit anti-GFP-biotin (1/25,000) allows detection of 25 ng GFP antigen (M.W.~27kDa).
Lanes 4-7: : PVDF membrane probed with rabbit anti-GFP-biotin (1/25,000) allows detection of Yes-GFP protein expressed using 1 and 10 ug of appropriately transiently transfected COS-7 cell lysates.
Note: the bands present at an apparent molecular weight of ~75 kDa and 150 kDa are endogenously biotinylated proteins (e.g. mitochondrial decarboxylases) commonly visualized when blotting with avidin-HRP conjugates.
1. McCabe, J.B. and Berthiaume, L.G. (1999) Functional roles for fatty acylated N-terminal domains in subcellular localization. Mol. Biol. Cell, 10, 3771-3786.
Zhang H et al. Multifunctional peptide-PEG intercalating conjugates: programmatic of gene delivery to the blood-brain barrier. Pharm Res27:2528-43 (2010).
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