Western blotting is usually time consuming. Sometimes, there are a number of proteins that need to be characterized per blot for an experimental run. However, sometimes these cocktails yield low quality blots. Non-specific bands or sequestration of the target bands are some of the encountered problems. What are possible reasons for these non-specific bands or the sequestration of the target band?
The problem of nonspecic binding still a problem with all immunoassays, specially with those indirect formats in which a secodary antibody is used. There are a number of articles addressing the problem of NSB in immunoassays. The only way to circumvent the problem is the optimization of the assay in a condition that produces the lowest level of NSB.
You can detect more than one target protein in immunoblot if the secondary antibody for the first antibody is the same. For example, you can detect various protein target with monoclonal antiboies and a secondary conjugate to the mouse Immunoglobulins.
For more information about NSB, please read our recent article (2012) in "Scandinavian Journal of Clinical and laboratory Investigations"
If MW of the target antigens are quite different, there is no problem at all to probed with a cocktail of two antibodies, as long as both antibodies have the same requirements for blocking buffer and time of incubation. The other consideration is do not mix antibodies when one of them has high backgrounds.
You just have to first determine what antibodies can be mixed without any problem. Based on my personal experience, I will advise
1. Do not mix antibodies for very highly expressed proteins with one with comparably very low expression.
2. When you have to mix, do not put all the antibodies into the dilluent and mix, instead, starting with the antibody with lowest protein expression, put the antibodies one at a time and mix well before putting the next.
Like Gabriela said, mw matters but not every time, I have successfully mixed beta actin and synaptophysin together and they work very well.
Another option is to cut your membrane if the mw are far enough apart and then incubate each cut section separately with the respective antibodies.
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