I am confused how much (amount) of antibodies and purified protein (antigen) to mix/adsorbed while making a pre-adsorption control for checking the specificity of antibodies by WB. And that, whether incubate it for 2 hours at room temperature or overnight at 4° C ??
Hi, Farman.
Make sure that you are mixing polyclonal Ab with purified matching antigen?
Isn't that tissue protein power?
Perhaps, clarify this.
Regards.
Dear professor Lee, thanks for your interest.
We are adsorbing polyclonal Ab with purified proteins. The protein we had in vitro expressed and purified not comes from tissues.
Hi, Farman.
You could first follow the basics of 1(Ab):1(Ag; molar ratio/molar ratio) theoretically saturated binding in the mixed solution. Because you want to have a fully-saturated, mixed solution to demonstrate Ab specificity, it is safe to put excess Ag for an over-saturated sol state. Let’s say that 2(Ag):1(Ab) molar ratio mix would give a probable, full saturation with excess free Ag in the sol. However, molecular mass difference should be taken care of. If the calculated difference in molecular masses of Ag is 5 times bigger, this would be another factor to be considered. Thus, the final ratio of Ab:Ag in the mix would be 2(Ag)x5:1(Ab; molar ratio/molar ratio) to adjust molar ratios in between Ab and Ag in a denatured buffer solution condition.
Make each sol at molecular concentration (x mM Ab and y mM Ag) separately. Then follow the order mentioned above.
Use any buffer containing 0.01% Tween 20 not to lose protein and prevent non-specific adsorption on the walls of tubes. Probably overnight incubation in cold room with a gentle rocking. Sterile or preventative conditions are essential, too by using a minimum sodium azide as a preservative after making and precipitating the mixture.
Best wishes.
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