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ADGP|ADP-glucose pyrophosphorylase介绍：

货   号：AS11 1739

中文名称：兔抗拟南芥腺苷二磷酸葡萄糖焦磷酸化酶多克隆抗体

英文名称：ADGP|ADP-glucose pyrophosphorylase

应用：western blot (WB)

规格：200 µl

价格：4440元

ADGP|ADP-glucose pyrophosphorylase简介：

application example

10 µg of total protein from Arabidopsis thaliana (1), Hordeum vulgare (2), Zea mays (3) total protein from all the samples were extracted with Protein Extraction Buffer PEB (AS08 300). Samples were diluted with 1X sample buffer (NuPAGE LDS sample buffer (Invitrogen) supplemented with 50 mM DTT and heat at 70 C for 5 min and keep on ice before loading. Protein samples were separated on NuPAGE 4-12% Tris-Bis gel (Invitrogen) LDS-PAGE and blotted for 1h to 1.5h on PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent (GE RPN 2125; Healthcare) or 5% non-fat milk dissolved in 20 mM Tris, 137 mM sodium chloride pH 7.6 with 0.1% (v/v) Tween-20 (TBS-T) for 1h at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1: 5 000 (in blocking reagent) for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, and then washed 1x15 min and 3x5 min with TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602) diluted to 1:25 000 in blocking reagent for 1h at room temperature with agitation. The blots were washed as above. The blot was developed for 5 min with TMA-6 (Lumigen) detection reagent according the manufacturers instructions. Images of the blots were obtained using a CCD imager (FluorSMax, Bio-Rad) and Quantity One software (Bio-Rad).2D gel electrophoresis was performed to exclude cross-reactivity of this antibody to Rubisco

WT-Col-0 Arabidopsis thaliana leaves were frozen in liquid N2 and soluble proteins were extracted in a buffer containing 50 mM HEPES, 5 mM NaCl and 10 mM MgCl2. 10 µg and 17 ug of native protein complexes were separated by non-reducing blue native PAGE (5-12,5 %) and then further separated in a second dimension by reducing SDS-PAGE (15% with 6M urea) and blotted 1h to a PVDF membrane using Höefer semi-dry blotter. Blots were blocked with 2 % milk in TTBS for 3h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 000 overnight in +4°C. The antibody solution was decanted and the blot was washed 3 x 5 min with TTBS at RT with agitation. Blot was incubated in secondary antibody (anti-rabbit IgG horse radish peroxidase conjugated, from Agrisera AS09 602) diluted to 1:25 000 in 1% milk/TTBS for 2h at RT with agitation. The blot was washed 3x5 minutes in TTBS and 1x5 min in TBS at RT with agigation and developed for 5 min with ECL according to the manufacturer\"s instructions. Exposure time was 30 seconds.Courtesy of Lauri Nikkanen, University of Turku, Finlandwestern blot on algal samplesTotal proteins (40 µg) from Chlamydomonas reinhardtii strain CC-124 (1), Chlamydomonas reinhardtii strain CC43-48 (STA6) (2) andPolytomella sp. Pringsheim 198.80 (3) were separated on a 5-12% acrylamide/6M urea SDS-PAGE and blotted onto nitrocellulose membrane. Blots were blocked in T-TBS (0.1% Tween in Tris Buffer Saline) containing 5% low fat milk, 1 h at room temperature (RT) with agitation. Blot was incubated in the primary antibody at a dilution of 1: 2 500 for 1h at RT with agitation. The antibody solution was decanted and the blot was washed twice for 15 min in T-TBS, RT with agitation. Blot was incubated in secondary antibody diluted to 1: 25 000 (goat anti-rabbit IgG, horse radish peroxidase conjugated, Agrisera, AS09 602), 1h at RT with agitation. The blot was washed as above and developed using the homemade enhanced chemiluminescence system (Durrant, Nature 346: 297, 1990). Images of the blots were obtained using a CCD imager (ImageQuant LAS4000). ​