Rabbit anti-Chicken IgY (H&L), HRP conjugated

Application example

(1), product number AS08 371S, 1 µg of total protein from samples such as Lycopersicum esculentum leaf (2), Nicotiana tabaccum leaf, (3),  Zea mays leaf (4), Hordeum vulgare leaf (5), Arabidopsis thaliana leaf (6) 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 keept on ice before loading. Protein samples were separated on 4- 12% Bolt Plus gels,  LDS-PAGE and blotted for 70 minutes to PVDF using tank transfer. Blots were blocked immediately following transfer in 2% blocking reagent  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: 10 000 (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 AS10 1489, Agrisera) 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 chemiluminescence detection reagent of extreme femtogram range, according the manufacturers instructions. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 30 seconds.

Purity of this preparation is > 95% based on SDS-PAGE. Antibody concentration is 1.0 mg/ml. Antibody is supplied in 10 mM sodium phosphate, 0.15 M sodium chloride, pH 7.2.1 % (w/v) B, Protease/IgG free. Contains 0.1 % (v/v) Kathon CG as preservative of bacterial growth.