This product is a suspension of affinity purified anti-GFP antibody (ab6556) covalently linked to sepharose beads. It is reactive against all variants of Aequorea victoria GFP such as S65T-GFP, RS-GFP, YFP, CFP, RFP, and EGFP.
The unit sold contains 25µg of affinity purified rabbit anti-GFP IgG cross-linked to 125 µl sepharose beads in a total volume of 250 µl buffer. The product is supplied as a 50% slurry to facilitate pipetting. Pipetting the slurry is facilitated when pipet tips are blunted by cutting 3-5mm off from the tip.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
We rountinely recommend using 20 µL of slurry per IP, which corresponds to 2 µg of IgG cross-linked to beads. this results in a clearly visible 10 µL bead pellet upon centrifugation. Unconjugated version: ab6556. FITC conjugated version: ab66180. Biotin conjugated version: ab69313.
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.
Lane 1 & 3: IP from COS 7 cells transfected with EGFP.N-Ras (48 kDa)
Lane 2 & 4: IP from untransfected COS 7 cells
Lane 1 & 2: IP using 15 ul of rabbit anti-GFP conjugated to sepharose beads (0.5 mg IgG per ml of beads)
Lane 3 & 4: IP using 15 ul of goat anti-GFP conjugated to sepharose beads (1 mg IgG per ml of beads)
Immunoprecipitation: COS 7 cell lysates containing 100 ug of total protein in 200 ul of 0.1% SDS-RIPA buffer with addition of complete protease inhibitor were used for each immunoprecipitation. Cell lysates were incubated with anti-GFP sepharose beads for 2 hours at 4oC with rocking. Beads were washed. Proteins were eluted with 1% SDS 50 mM HEPES (pH 7.4) at 80oC (15 min). Half of the IP sample was loaded on each lane of 10 % SDS PAGE gel and gel was processed for Western blotting/ECL.
Western blot: Primary antibody: monoclonal mouse anti-GFP (ab291) at 0.2 ug/ml in 5% non-fat milk/TBS-T; 1 hour incubation at room
Chou YT et al. Identification of danthron as an isoform-specific inhibitor of HEME OXYGENASE-1/cytochrome P450 reductase interaction with anti-tumor activity. J Biomed Sci25:6 (2018).
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