Anti-DPE2 | 4-alpha-glucanotransferase

Arachis hypogaea, Brachypodium distachyon, Brassica napus, Cannabis sativa, Glycine max, Gossypium sp., Hordeum vulgare, Malus domestica, Manihot esculenta, Medicago truncatula, Nicotiana tabacum, Oryza sativa, Pisum sativum, Populus sp., Ricinus communis, Solanum lycopersicum, Solanum tuberosum, Sorghum bicolor, Spinacia oleracea, Theobroma cacao, Triticum sp., Vitis vinifera, Zea mays

Samples:
1 - Protien Ladder
2-4 - Columbia-0, Arabidopsis thaliana wildetype
5-7 - fzl-3 mutant (overexpressor)
8-10 - dpe2-5/phs1b mutant (knockout)

40 µg of total protein extracted freshly from Arabidopsis thaliana leaves was loaded/well. Exact buffer components were: 9% gel was used, seperating gel consists of (0.75M Tris/HCl pH 8.8, 10% SDS, Rotiphorese GEL 30, 10% ammonium persulphate and TEMED) and stacking gel with (0.5 M Tris/HCL pH 6.8, 10% SDS, Rotiphorese GEL 30, 10% ammonium persulphate and TEMED) and running buffer consisting of (Tris, glycin and SDS) denatured with 3x SDS sample buffer to the crude extract i.e (10 µl SDS buffer to 20 ul crude extract) at 95 °C for 5 min at 1200 rpm and centrifuged at RT for 15 min at 14000g. western blot Samples were transferred in the cold transfer buffer (50 mM Tris, 150 mM Glycin, 20% methanol and 0.02% (w/v) SDS and blotted for 50 min in 40V and 250 mA. nitrocellulose (pore size of 0.2 um), using wet transfer in the cold. Blot was blocked with 5 % milk for 1h/RT with gentle agitation. Blot was incubated in the primary antibody at a dilution of 1:500 for 1h/RT with gentle agitation (diluted in milk blotting buffer). The antibody solution was decanted, and the blot was then washed 6 times for 5 min in TBS-T at RT with gentle agitation. Blot was incubated in matching secondary antibody (anti-rabbit IgG horse ALP conjugated, AS09 607, Agrisera) diluted to 1:1000 in for 30 min-1h/RT with agitation. The blot was washed 6 times and 5 min each in TBS-T as above and developed with BCIP/NBT for 15 minutes.

Courtesy of Biopolymer Analytics, University of Potsdam, Germany