AgriseraECL BrightEnhanced (100 ml)
AS16 ECL-100 | luminol based, two component, enhanced and stabilized chemiluminescence substrate with femtogram detection levels
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|Expected | apparent MW|
|Not reactive in|
1 µg of total protein from Arabidopsis thaliana leaf (1), Hordeum vulgare leaf (2) 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 (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 affinity purified anti-PsbA primary antibody at a dilution of 0.1 µg/ml (Standard Protocol, right panel) (in blocking reagent) for 1h at room temperature with agitation or added to Agrisera IncuBlocker (rabbit) rapid western blot reagent and incubated for indicated amount of time together with appropriate secondary antibodies included in IncuBlocker (left panel).
Following standard protocol: 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 (goat anti-rabbit IgG horse radish peroxidase conjugated, recommended secondary antibody AS09 602, 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 TMA-6 (Lumigen) detection reagent 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.
Do not add any protein or HRP enzyme to reagent solution.
Optional: wash your tube, microtiter plate or membrane with 0.2 M solution of Sodium Phosphate, Dibasic, in Deionized Water. (Initial pH may be approximately 9) Bring the pH down to 8.4 with the slow addition of a 0.2M solution of Sodium Phosphate, Monobasic, in water.
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