Doing WB from scratch for the first time. Need some info. - (Dec/30/2010 )
Hi, I will be doing a western blot in the next couple of weeks from scratch but I don't really know how to do it, specifically choosing the antibodies. I have done a western blot before but I was handed everything and just had to follow the protocols. I did a search on the forum and did not find much help. Can you guys please point me to the right direction about how to choose my antibodies?
Also, I have a question about the antibodies. Do primary antibodies have universal fragments (where the secondary antibody binds to)? Not sure if I worded that properly. Basically what I'm asking is, will a primary antibody from bio-rad be compatible with a secondary antibody from sigma aldrich?
when you chose antibodies you first decide which is your protein of interest.
When there are more antibodies available, you check whether they recognize your species (e.g. human cell lines vs. mouse...) and how specific they are. And often you then prefer monoclonal antibodies if possible.
The primary antibodies are named after the species they are produced in , very often rabbit or mouse. This means, when you have a mouse primary antibody it is produced in mice, it does not mean that it is automatically against a mouse protein.
The secondary antibodies are directed against IgGs of this hosts.
When you have a primary antibody produced in mice as secondary antibody you have to chose one against mouse, for example goant anti mouse - means it is directed against mouse IgG and was produced in goat.
Here it does not matter if this antibody is produced by the same company as the primary one is.
You should also pay attention to the application that the primary antibody is meant for. All the companies will tell you whether the antibody can be used in Western blot and give you suggested starting dilutions. This is important because WB usually uses denatured/reduced protein (unless you run a native--no SDS--and/or reduced--no DTT, beta-mercaptoethanol--gel), which is a different antigen than a native protein. So an antibody for flow cytometry may or may not work in Western blotting.
Most primary antibodies will be whole, meaning they have an Fc and Fab fragments. So in this case you don't need to worry about the secondary recognizing a specific fragment. A secondary antibody which recognizes Fab, Fc or H+L will bind a whole antibody, so long as it's the right host. As the person said above, if your primary antibody is mouse-anti YFP then you want a conjugated goat (or rabbit or sheep, etc) anti-mouse. And definitely antibodies from different companies will be compatible, so long as you have your combinations right.