GFP | Green Fluorescence Protein (total IgY)
AS15 2998 | Clonality: Polyclonal | Host: Chicken | Reactivity: native and recombinant GFP
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highly purified native GFP protein derived from Aequorea victoria, UniProt: P42212
20 ug of Arabidopsis thaliana Columbia-0 overexpressing GRP8-GFP leaf protein extract (1), 70 ug of Arabidopsis thaliana Columbia-0 overexpressing GFP-SnRK2.4 leaf protein extract (2), 50 ug of Nicotiana benthamiana transiently expressing GFP-SnRK2.4 leaf protein extract (3), 50 ug of Nicotiana benthamiana transiently expressing SnRK2.4-GFP leaf protein extract (4). Mark: MW markers: PageRuler Prestain Protein Ladder from Thermo Fisher Scientific (#26616) The total protein extracts were freshly prepared from A. thaliana leaves with extraction buffer 1 (samples 1&2) or N. behtamiana leaves with extraction buffer 2 (samples 3&4). The extraction buffer 1 contained: 20 mM Tris-HCl pH 7.5; 2 mM EDTA; 2 mM EGTA; 50 mM β-glycerophosphate; 250 mM sucrose; 10 mM Na3VO4; 1% Triton X-100; 150 mM NaCl; 10 mM DTT; 1 mM PMSF and 1 x Complete Protease Inhibitor Cocktail (Roche). The extraction buffer 2 contained: 100 mM HEPES, pH7.5; 5 mM EDTA; 5 mM EGTA; 10 mM DTT; 1 mM Na3VO4; 10 mM NaF; 50 mM b-glycerophosphate; 10 mM pyridoxal 5-phosphate; 10% glycerol and 1 x Complete protease inhibitors (EDTA-free, Roche). The extraction buffers were added to the powdered material in a 1:1 v:v ratio. Samples were incubated on a rotator for 30 min at 4°C and then centrifuged for 30 min, 12 000 rpm at 4°C. Supernatant was transferred into new tubes and protein concentration was measured using Bradford Protein Assay. Samples were then denatured with 3x Laemmli sample buffer for 5 min in 95°C. Next samples were separated on 10 % SDS-PAGE stain-free gels (Bio-Rad) and blotted overnight to PVDF membrane using wet transfer. Blot was blocked with 3% milk and 0,1% Tween 20 in TBS for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 for 1,5h/RT with agitation in 3% milk and 0,1% Tween 20 in TBS solution. The antibody solution was decanted and the blot was rinsed briefly, then washed 5 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (rabbit anti-chicken IgG horse radish peroxidase conjugated, AS10 1489, Agrisera) diluted to 1:10 000 in for 1h/RT with agitation. The blot was washed as above and developed for 1 min with Pierce ECL Western Blotting Substrate. Exposure time was 5 min (short exposure)/ 10 min (long exposure). Additionaly blot was briefly rinsed with water and developed for 1 min with chemiluminescent detection reagent. Exposure time was 1 min.
Courtesy of Katarzyna Szymańska, Institute of Biochemistry and Biophysics PAS > Plant Protein Phosphorylation Laboratory, Warsaw, Poland
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.
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