antibody incubation on western blots - difference between 4°C and RT (Dec/11/2007 )

hallo,

don´t know if this is a stupid question:
normally i incubate by 1st antibody at 4°c overnight.
what will happen if i incubate it on RT overnight? will it worsen the specificity of binding?

thanx

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4 C allows binding more easily than RT (less dissociation).

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Then you should also do the next steps at 4°C ?

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I don't think you have to continue the whole protocol at the same temp; when I do a western I use a combination of temps. often it depends on my time - 4C just slows down the reactions that take place at RT, both good and bad

if you chose to do subsequent steps at 4C, you might be likely to have to increase your incubation times

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O.N. incubation at 4°C is not necessary for 2nd Ab but may increase unspecific binding;

O.N. incubation at RT may damage the antibody; this is interesting if you like to re-use the antibody...

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Usually I do 1-2 hours at RT.
Eventually, I do at 4°C when I don't want to stay late in the lab, and continue the experiment the day after. but there is no additional benefit for this kind of incubation. At 4°C you have less dissociation, but it's no more true when you incubate then the next steps at RT.

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to answer your question : there is no benefit to increase incubation time up to ON at room temperature.
The antibody should bind within one hour. If you incubate longer, you will only have more background, and eventually antibody degradation.

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And the antibody is in blocking solution. Fungi love blocking buffers, so if you want to reuse it ...

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4'c slows down the reaction. if often means an overnight incubation will give you a nice strong band. overnight at RT might give you multiple bands.

THOUGH... we did have one antibody that did not work. someone started a western on a friday, got sick, and didn't come back to the antibody till monday morning. the western worked perfectly. turned out that antibody needed the extra time.

there are things you can add to the antibody solution if you reuse it. sodium azide springs to mind. that seems to stop fungi growth.

V

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We re-use our primary antibodies too. After the first usage I add sodium azide (0.05% final) and keep in the fridge.

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