how to select the proper antibody - (Dec/25/2007 )
I would like to purchase an antibody. One manufacturer offers different choices of that antibody in terms of which part of the protein it recognizes (epitope): C-terminus, N-terminus or full length, while all other criteria for that antibody are the same; i.e. applications, origin, species.
I want to use that antibody for WB and IP of my target protein, so based on the epitope, which antibody should I select?
The company's catalog will tell you whether the antibody is suitable for both WB and IP.
If not, you may wish to call the technical service.
Some antibody may only suitable for WB, but not IP.
Try a reputable antibody company, such as cell signaling...
Only try Santa Cruz, if your required antibody is not available from other company.
Santa Cruz does not test every single antibody clone.
Hope this may help.
the antibody that recognizes different epitopes (c-terminal, n-terminal and full length) on the same protein is suitable for both WB and IP, so my question is still unanswered. Does the epitope make a difference?
yes, the epitope makes a difference. for western blot it does not matter but for ip you need a natively exposed epitope.
one of the terminus-specific antibodies may be okay but it is not likely that both will.
the antibody that is made against full length is probably the best bet (especially if it is polyclonal).
If all the antibodies that you checked can be used for both IP and WB (and maybe for other applications also), and the only difference is with epitope, then the choice of the Ab will depends largely on the purpose of your exp.
If you only want to do WB or IF, then it does not matter which Ab you are using. You may want to consider to use one Ab from mouse, for example, for WB, while you also use another Ab from mouse, for IP, so that there will be less chance of getting additional bands from IgG chains.
If you want to do IP, and then check for interacting proteins, then you have to consider more carefully. Because the Ab that you use for IP could compete with the interacting proteins (which you want to check for) if the binding domains (the epitopes) are overlapped. This could be a bad thing if you want to pull-down the interacting proteins. So make your choice carefully.
Before ordering any antibody, check literature to see if they have been used to get an idea.