We recently started using a Licor Odyssey Fc machine and got great results with the blocking buffer that Licor provides with the machine. However, we have used almost all of it and it is really expensive...so we tried our old recipes we used for our kodak imager/chemi that is simply non fat dry milk, tween, diH20 and TBS. Unlike previous results with the Licor BB (pure black background with distinct bands) my bankground was extremely red. I started researching other alternatives such as fish gelatin etc and came across soy milk...anyone have any experience with this???
Thank you! We are using nitrocellulose though...does thay make any difference
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3383026/pdf/nihms368359.pdf
"Soymilk: an effective and inexpensive blocking agent for
immunoblotting"
I used LiCor Odyssey for 0.2um PVDF membranes blocked and incubated with primary antibody in 2.5% BSA V, secondary in 2.5% milk. The background was just fine. I smell something fishy in the washing steps and it is not the skin gelatine!
After thousands of westerns I came to conclusion that there are only few antibodies which don't work with BSA. Major factors affecting antigen - antibody reaction are ionic strength and pH of the solution.
I have attached the image showing four different blocking conditions: 5% Milk in TBS, 5% BSA in TBS, Odyssey Blocking Buffer/TBS (1:1) and Odyssey Blocking Buffer /TBS (1:2) on nitrocellulose membranes probed with mouse anti-actin and goat anti-mouse800CW. They were all really nice with no background, however, the actin bands were weaker in Milk and when tested with the red channel (700nm), Milk gave rise to autofluorescence.
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