What is the reason to use a secondary antibody if we can just use the primary antibody linked with a reporter enzyme?
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Usually the secondary antibody will have a reporter function. This builds flexibility in Western blotting in case the reporter system isn't working, or if the secondary antibody doesn't work.
It's very costly to produce a primary antibody against an epitope (the thing the antibody will recognize) AND also tag the primary antibody with a reporter function. After taking time to produce and purify antibodies that will specifically recognize an epitope, it would be risky to perform the chemistries needed to also engineer those antibodies with a reporter function. In doing so, you risk altering the behavior of the antibody so that it no longer recognizes the epitope as efficiently or at all.
It's much easier to use an antibody to probe for the epitope of interest. Then depending on what organism made the primary antibody, you can get a second antibody with a reporter function to bind specifically to the primary antibody. For example, if you made the primary antibody using mice, then the second antibody will need to recognize mice antibodies. Again, it's easier to make an antibodies that recognize general mice antibodies rather than a specific mouse epitope.
The flexibility comes in when you have problems having the secondary antibodies recognize the primary antibodies. In the above example, say you used secondary antibodies made in rabbits. If those rabbit anti-mouse antibodies don't work too well, then all is not lost. You can get anti-mouse antibodies made from goat or horse or monkey, all with different reporter systems (in case the reporter systems don't work either). Thus, the primary antibody will still function well, but you tweak the secondary antibody to get the best signal out of the blot.
The other reason to use a secondary antibody is to get a better signal. Imagine the primary antibody binding to a single epitope (or two). So that's one molecule on the blot. If only that antibody glowed, the signal would be very weak. But if you use a secondary antibody against the primary, then several of the secondary antibodies can bind to the primary antibody (especially if the secondary antibodies are polyclonal, the different secondary antibodies will bind to different parts of the primary antibody) with each of the secondary antibodies reporting a signal. So the entire complex would be made up of one or two epitopes, one primary antibody and perhaps four secondary antibodies each of which will report a hit. Using secondary antibodies, the signal would be four times stronger than just a single primary antibody.
It's very costly to produce a primary antibody against an epitope (the thing the antibody will recognize) AND also tag the primary antibody with a reporter function. After taking time to produce and purify antibodies that will specifically recognize an epitope, it would be risky to perform the chemistries needed to also engineer those antibodies with a reporter function. In doing so, you risk altering the behavior of the antibody so that it no longer recognizes the epitope as efficiently or at all.
It's much easier to use an antibody to probe for the epitope of interest. Then depending on what organism made the primary antibody, you can get a second antibody with a reporter function to bind specifically to the primary antibody. For example, if you made the primary antibody using mice, then the second antibody will need to recognize mice antibodies. Again, it's easier to make an antibodies that recognize general mice antibodies rather than a specific mouse epitope.
The flexibility comes in when you have problems having the secondary antibodies recognize the primary antibodies. In the above example, say you used secondary antibodies made in rabbits. If those rabbit anti-mouse antibodies don't work too well, then all is not lost. You can get anti-mouse antibodies made from goat or horse or monkey, all with different reporter systems (in case the reporter systems don't work either). Thus, the primary antibody will still function well, but you tweak the secondary antibody to get the best signal out of the blot.
The other reason to use a secondary antibody is to get a better signal. Imagine the primary antibody binding to a single epitope (or two). So that's one molecule on the blot. If only that antibody glowed, the signal would be very weak. But if you use a secondary antibody against the primary, then several of the secondary antibodies can bind to the primary antibody (especially if the secondary antibodies are polyclonal, the different secondary antibodies will bind to different parts of the primary antibody) with each of the secondary antibodies reporting a signal. So the entire complex would be made up of one or two epitopes, one primary antibody and perhaps four secondary antibodies each of which will report a hit. Using secondary antibodies, the signal would be four times stronger than just a single primary antibody.
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A secondary antibody is an antibody that binds to primary antibodies or antibody fragments. They are typically labeled with probes that make them useful for detection, purification or cell sorting applications.
Secondary antibodies may be poly clonal or monoclonal, and are available with specificity for whole GI molecules or antibody fragments such as the Fc or Fab regions.
Secondary antibodies may be poly clonal or monoclonal, and are available with specificity for whole GI molecules or antibody fragments such as the Fc or Fab regions.