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Primary antibody incubation length - (Jul/12/2009 )

Hi Guys,

I have a quick question. How long do you all typically incubate your primary antibodies on PVDF transferred proteins for western blot purposes? The reason I ask is that I was accustomed to doing an overnight incubation at 4C in a roller but the new lab I work for actually does theirs at room temp for only an hour. Higher antibody concentrations than what I'm used (1:500) to but it seems to work for them. Is there any reason why overnight would be preferable to just 1 hour incubation length and is there any particular reason that most protocols call for a 4C incubation rather than room temp?

Also I know this second question depends a lot on the target quantity but how much protein do you folks usually load onto your gels?



The difference is rate of reaction, at 4 degrees, potentially the reaction will proceed slower, but it will also be less specific, while at room temperature, the reaction should be faster (nearly twice as fast if I recall correctly) and more specific, but the antibody will also degrade more. Some antibodies are quite unstable too, so even a short time at room temperature will kill it.

The reason for doing overnight is probably one of two things:

1)4 deg C will often get a poor affinity, low concentration antibody to work with overnight incubation, when it might not work at room temp for a much shorter incubation.
2) Historical, it fits in nicely with running, transferring, blocking being one day, then primary overnight... With modern methods, you often can do the whole proceedure in one day.

A third thing that could be the case, is that the commonly used milk powder block/antibody diluent is pretty prone to going off, 4 deg will delay this quite a bit.


laziness - oh i cant be bothered waiting an hour, just put it in the cold room overnight, i'll sort it tomorrow




bob1 on Jul 12 2009, 04:51 PM said:

The difference is rate of reaction, at 4 degrees, potentially the reaction will proceed slower, but it will also be less specific

Yes and no. The reaction will proceed slower but will also likely proceed with more specificity. If you think about the molecular dynamics involved, antibodies have high binding affinity towards their target epitope. Non-specific binding, however, is a much lower affinity. At 4 degrees the total energy of the system is reduced. This means that while the specific binding affinity of the antibody to your target protein is slightly reduced it is still effective. Conversely, the non-specific interactions are greatly reduced or completely removed because there isn't enough energy to bind well.

As mentioned by Bob, it can also be used for low-affinity or poor antibodies to give them a better chance of binding. Some antibodies (such as antibodies directed against phospho-proteins) are notorious for requiring incubation overnight.

Our lab routinely does either 2 hour room temperature incubations or overnight 4 degree incubations. Without question, the overnight incubations always look better. However sometimes you are in a rush and the 2hr room temp is more convenient.