Why antibodies binds specifically to their targets in 4 oC rather than room temperature. What exactly happens in this over night incubation?
First, longer incubation demands more stability of the solution overall (ie. evaporation), not only the abs. That is one reason, i think. Other reason is mighbe that there are abs where hydrophobic interactions are important so lower temp is preferable (means stronger interaction for that type of ab only, of course).
I have read some troubleshoot said that RT incubation could induce more background than 4C. This also depends on your interested protein. Some protein is very high expression and 1-2h RT incubation is enough but some protein is very low affinity, so it works better at 4C overnight. I would say just try it, and stick with the way that works the best for each protein.
Please look at this equation: Ag + Ab = AgAb + Delta G
it means this reaction is releasing energy and if you remove this energy, you will have more products. However, you have to wait a longer time. In RT you elevate the kinetic or the molecule movements of the reaction with shaking and temperature. Therefore, it depends on your situation to choose one, although researchers prefer not to waste time.
Most antibodies (Ab) of respectable avidity will reach Ab/antigen (Ag) equilibrium in ~2 hours at 22 deg C and less time at 37 deg. Low avidity Ab can take longer, perhaps, overnight, although mild shaking should speed things up, as Dr. Rezaee indicated. However, if a 4 deg condition is really important, it probably means that the Ag is unstable at room temperatures, and low temp is needed to prevent Ag denaturation or proteolysis. There are also some naturally-occurring human cold-reactive antibodies, but this is not generally seen. Often, overnight incubation is prescribed for human convenience, and 4 deg is used to minimize evaporation or denaturation of reactants. If such long incubation times are not convenient for you, I would not hesitate to try 2 hrs at R.T. If an Ab really binds better at 4 deg, and this is not due to Ag stability issues, it is purely fortuitous that low temperatures enhance immune complex formation, especially if the Ab was produced in vivo (i.e, at 37 deg).
All the reasons given above are true and its best explained depending on the type of antibody and how it reacts with its target. For low affinity ones, overnight at 4 deg C is preferable to allow enough time for the reaction while maintaining the stability of the overall reaction solution. From my experience, I used some antibodies both overnight at 4 deg as well as at RT (22 to 25 deg) for 3 to 4 hrs and both the situations gives same results.
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