1. Is 2view™ suitable for both nitrocellulose and PVDF membranes?
Yes. 2view™ has been tested on nitrocellulose and PVDF membranes from different manufacturers (Expedeon, NXA19020 and NXA29320 ; Invitrogen, LC2001; Biorad, 1620117; Merck Millipore, IPVH00010) and data showed reproducible results as well as high signal-to-noise ratio.
2. Is 2view™ suitable with different blocking agents other than BSA?
Yes. 2view™ is suitable with the most commonly used blocking agents, such as non-fat dry milk (3-5%), casein (1%) and fish skin gelatin (1%). Also, it is compatible with 0.15-0.5M NaCl and 0.05-1% Tween20®. Non-fat dry milk and casein are not recommended when using a biotin-avidin interaction as milk contains large and variable amounts of biotin.
3. What is the recommended dilution range for the primary antibody?
Primary antibody dilution range is often between 1:500 to 1:5000 dilution from a 1 mg/mL stock. However, this may not be true for all antibodies and you should also consider the range suggested in the supplier datasheet.
4. What is the recommended dilution range for 2view?
2view™ has been optimized for a 1:25 dilution. However, 2view™ will work in a dilution range from 1:10 to 1:50 but this could require further wash steps and longer incubation time, respectively.
5. Can diluted 2view™ in blocking buffer be re-used?
Yes, it can be re-used up to 3 times; store at +4°C for up to 2 weeks. It is recommended not to re-use 2view™ on a membrane probed with a different primary antibody.
6. Can other LumiBlue ECL solutions from Expedeon (i.e. ECL Extended, ECL Extreme) be used instead of ECL Pico to increase sensitivity?
Yes, however the primary antibody concentration must be optimized (higher dilution) to keep the background low.