For western blotting, the primary antibody incubation time is very crucial. And, the incubation time depends on the binding affinity of the antibody and abundance of protein. I would like to know how can I determine the perfect incubation time based on the binding affinity of primary antibody?
The association between antibody and antigen is actually very quick, as you can see when doing kinetic measurements, for instance in Biacore. Of course, it varies from one antibody to another, depending on their kon values, but in general it takes place in times that are much lower than the commonly used incubation times in immunochemical techniques (one-two hours at RT or 37 degrees). Actually, these commonly used times represent a huge excess to guarantee binding without concerns. So I would not say incubation time is very crucial.Some people prefers to do the incubation at 4 degrees overnight. At this temperature everything is slower, but in certain cases the background is reduced.
What is strictly dependent on the affinity is the required concentration of the primary antibody. Dissociation of the antibody can also be quick (depending on koff). If you have a low affinity antibody the equilibrium between association and dissociation can be reached at low binding levels (despite you increase the incubation time). If you increase the concentration of the antibody you would favor the formation of the antigen-antibody complex, having higher signals. Often an excess of primary antibody in the order of ug/ml is used (if avaliable) independently of the affinity to make sure that its enough. If your antibody has a very low affinity, you would have to increase the concentration as much as possible, as long as it doesnt result in a high background. You can titrate the antibody in blots containing positive and negative samples to determine a good working concentration.
The association between antibody and antigen is actually very quick, as you can see when doing kinetic measurements, for instance in Biacore. Of course, it varies from one antibody to another, depending on their kon values, but in general it takes place in times that are much lower than the commonly used incubation times in immunochemical techniques (one-two hours at RT or 37 degrees). Actually, these commonly used times represent a huge excess to guarantee binding without concerns. So I would not say incubation time is very crucial.Some people prefers to do the incubation at 4 degrees overnight. At this temperature everything is slower, but in certain cases the background is reduced.
What is strictly dependent on the affinity is the required concentration of the primary antibody. Dissociation of the antibody can also be quick (depending on koff). If you have a low affinity antibody the equilibrium between association and dissociation can be reached at low binding levels (despite you increase the incubation time). If you increase the concentration of the antibody you would favor the formation of the antigen-antibody complex, having higher signals. Often an excess of primary antibody in the order of ug/ml is used (if avaliable) independently of the affinity to make sure that its enough. If your antibody has a very low affinity, you would have to increase the concentration as much as possible, as long as it doesnt result in a high background. You can titrate the antibody in blots containing positive and negative samples to determine a good working concentration.
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