Agrisera ECL kit (Bright/SuperBright)
This product can be purchased in 3 different volumes:
AS16 ECL-SN-10, 10 ml. Trial size limited to one per customer
AS16 ECL-SN, 100 ml
AS16 ECL-SN-1L, 1L
Choose the appropriate volume in the drop down menu to the right
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It is a ready to use 2 component system with low background and superior signal to noise ratios and moderate to highest sensitivity offered by:
Agrisera ECL SuperBright
Extreme low femtogram detection
Agrisera ECL Bright
Low pico to mid femtogram detection
Depending on the abundance of the protein under investigation, different sensivity levels of chemiluminescent reagent used for detection can be applied, as compared in this example.
10 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 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 (AS05 084A) at a dilution of 1: 40 000 from 1 mg/ml stock.
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 1:20 000 in blocking reagent. Agrisera ECL SuperBright or Agrisera ECL Bright were used respectively, for detection. Images of the blots were obtained using a CCD imager (VersaDoc MP 4000) and Quantity One software (Bio-Rad). Exposure time was 60 seconds, enhanced.
- Store reagents A and B in the darkness at 4-8°C.
- Mix equal volumes of reagent A and B (chemiluminescent substrate) in a clean container and equilibrate to room temperature 30 minutes before use.
- Prepare your membrane prior addition of chemiluminescent substrate, by a wash with the buffer used in your protocol (PBS or TBS or TBST-T). This will allow to remove any background prior to substrate contact.
- Optimal visualization is obtained up to 20 minutes after substrate contact. Incubation for 2-5 minutes is usually optimal.
- Remove excess substrate by filter paper.
- Cover blot with clear plastic wrap or sheet protector and expose either with x-ray film or CCD camera.
For best results clean containers and high quality water has to be used.
HS code for this product is: 3822.00.0002.
Related products: Agrisera ECL kit (Bright/SuperBright)
This product can be purchased in 3 ...
This product can be purchased in 3 different volum...