Generally, we incubate the blocked membrane with the primary antibody, wash away the unbound primary antibody, then incubate with the secondary antibody which binds to the primary antibody, and detect the protein of interest on the membrane. Since the primary antibody binds its epitope with its variable domain, whereas; the secondary antibody binds with its constant domain, there should be no reason that mixing these two antibodies together will interfere with the binding. Can we incubate the blocked membrane with both primary and secondary antibodies at the same time for Western Blotting ? If it works, it will save time.
This is a great question.
Think about what would happen with both antibodies together in solution. Because each 2o antibody can bind to 2 1o antibody molecules and each 1o antibody molecule has multiple potential sites of interaction with 2o antibody molecules, you would end up forming an enormous complex of 1o and 2o antibody molecules, which would probably precipitate instead of binding to the antigen on the membrane.
It is possible to dispense with the 2o antibody if you make an enzyme conjugate of the 1o antibody, but there is a reduction in sensitivity because you don't get the amplification of multiple 2o antibodies binding to one 1o.
This is a great question.
Think about what would happen with both antibodies together in solution. Because each 2o antibody can bind to 2 1o antibody molecules and each 1o antibody molecule has multiple potential sites of interaction with 2o antibody molecules, you would end up forming an enormous complex of 1o and 2o antibody molecules, which would probably precipitate instead of binding to the antigen on the membrane.
It is possible to dispense with the 2o antibody if you make an enzyme conjugate of the 1o antibody, but there is a reduction in sensitivity because you don't get the amplification of multiple 2o antibodies binding to one 1o.
Hi Shweta
I think it is possible. Though I haven't tried putting 1st and 2nd together for incubation, I've done incubating two 1st abs at the same time and it worked. However, one thing I realised after trying so many times to "save time" is that: doing separately makes your life easier when analysing the "bad" results (e.g strong background, nonspecific binding, missing bands, unexpected trends). Because if something goes wrong, it is hard to tell what the cause was: 1st ab, 2nd ab, or the interaction of the two? So if you are not in a rush for presentation or publication, I think it's better to do one by one.
Of course, you can try by yourself to see what happens.
Cheers,
Long
That is a good question, never considered it.
Some Immunoprecipitation protocols also incubate antibody and epitope in solution before adsorption to a solid substrate (the beads).
But in the case of a Western blot: I think too many secondary antibodies are titrated away when they are incubated together with the primary. Because in a standard Western blot protocol, after incubation with the primary Ab, the non-bound Ab's are washed away before incubation with the secondary Ab. If you omit this step, I think a lot of your secondary Ab's will be 'captured' by the free floating primary Abs before they can bind to the primary Abs bound to the membrane.
As far as the suggestion that a big complex might be formed: I think coagulates might be formed when the secondary is a polyclonal or of the IgM type. But anyway the result will be the outtitration of secondary Ab's.
So I think it is possible, but you will have a (much) lower signal. Maybe when you increase the concentration of secondary it might work.
I think the best answer to this question can be given by nature: try it!
More of this, the time of incubation is different for the 1st and the 2nd antibodies. Sometimes it is better to incubate the membrane with the 1st antibody over night at 4 dgrs for a proper result. If you put the secondary antibody a lot of time with the membrane, or more concentrated, it would be possible to bind unspecific and to obtain many unspecific bands or background. And it is right that the TBST washes between the antibodies have a great importance in obtaining a good and clear result of WB. So the protocol was established very well and for certain reasons in the way to put the 1st and the 2nd antibodies separately.
I think this will work good if primary antibody is highly specific. Moreover you need proper TBST washing followed by incubation. Some antibodies are so good to give good signal after even one hour of incubation at room temperature. So I think this condition depends on specificity of antibody.
It dose not seems good ,because you save time but instead you reduce ...
This will make poor band of your primary antibody and that's why we must wash the the membrane in between the primary and the secondary antibodies, so even with small traces can cause interaction between the two and poor images.
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