GFP | Green Fluorescence Protein
AS20 4443 | Clonality: Polyclonal | Host: Rabbit | Reactivity: Green Fluorescence Protein (GFP)
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194 µg/well of total protein extracted freshly from tobacco with write exact buffer components (50mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% Glicerol, 1 mM DTT, 1X Pefablock) and denatured with SDS 2% and 0.1% Bromophenol blue (Laemmli buffer) at 95°C for 5 min.... were separated on 12 % SDS-PAGE and blotted 2hs to PVDF/nitrocellulose (pore size of um), using wet transfer. Blot was blocked with 5% milk for 1h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 10 000 in TBS-T ON/4°C (with or without milk) with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 10 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, AS09 602) diluted to 1:20 000 in for 1h/RT with agitation. The blot was washed as above and developed for 2 min with Agrisera ECLSuperBright with name and supplier. Exposure time was 60 seconds.
Courtesy of Delfina Gagliardi Instituto de Agrobiotecnología del Litoral (IAL), Argentina
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