GFP | Green Fluorescence Protein (affinity purified)
AS15 2987 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Native and Recombinant GFP
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Highly purified native GFP protein derived from Aequorea victoria, UniProt: P42212
Detection of YFP-tagged Lamin A in the nucleus of mouse fibroblasts using GFP | Green Fluorescence Protein (affinity purified) antibodies. Fixation and permeabilization was performed with methanol at -20°C for 10 min. a) Indirect immunofluorescence labeling of YFP-lamin A with anti-GFP primary antibody (dilution 1: 500) as primary antibody and detected by TRITC-conjugated goat-anti-rabbit as secondary antibody.
b) Green fluorescence image of YFP-tagged Lamin A, which is incorporated into the nuclear lamina and tubular structures that penetrate into the nucleus.
c) DAPI staining of nuclear DNA.
d) Merged images
Protein extracts were obtained from Arabidopsis thaliana seeds (producing atLEA4-5 fused to GFP; other lanes contain GFP-6H loaded in indicated amounts). Following extraction buffer was used: 0.7 M sucrose, 0.5 M Tris-base, 30 mM HCl, 50 mM EDTA, 0.1 M KCl, 2% β-mercaptoethanol, 12 mg/ml poly-vinyl-poly-pyrrolidone (PVPP). This buffer was complemented with 2mL of equilbrated phenol before extraction. Samples were centrifuged and the protein phase was recovered; the extracted proteins were precipitated with 0.1 m ammonium acetate dissolved in methanol, centrifuged and the pellet washed with cold (-20 ℃) 80% acetone. The protein pellet was dissolved in SDS-solubilization buffer (1% CHES, 2% SDS, 2% ß-mercaptoethanol, 10% glicerol). Thirty 𝜇g of seed protein extract was denatured with Laemmli buffer at 95 °C for 5 min and proteins were separated on 15% SDS-PAGE and blotted 1.5 h to nitrocellulose membrane in a liquid transfer system. Blots were blocked with 2% skim milk ON at 4°C with agitation. After rinsing with TBS, blots were incubated in the primary antibody at a dilution of 1:10 000 (anti-GFP) for 3h at 4°C with agitation. The antibody solution was decanted and the blot was rinsed briefly, then washed once for 15 min. and 3 times for 5 min in TBS-T at 4°C with agitation. Then, blots were incubated in secondary antibody (anti-rabbit IgG horse-radish peroxidase conjugate) diluted to 1:25 000 in for 1h at RT with agitation. The blot was washed as above and developed for 2 min with SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific. Exposure time was for 30 seconds.
Courtesy of Dr. Alejandra Covarrubias, UNAM, Mexico
Minimal cross-reactivity with E.coli proteins.
GFP (Green fluorescent protein) was originally identified in photo organs on jellyfish Aequorea victoria. It is a naturally fluorescent protein which emits green light at a maximum wavelength of 509 nm when excited by blue or UV light. It is extensively used in laboratory as a reporter molecule to label and study cellular and subcellular proteins in living cells using a wide range of applications. Antibodies to GFP protein are used in immunoblotting and ELISA. GFP protein has molecular weight of 27 kDa.
Kulich et al. (2018). Deubiquitinase OTU5 affects Root Responses to Phosphate Starvation. Plant Physiol. 2018 Jan 4. pii: pp.01693.2017. doi: 10.1104/pp.17.01693.
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