I need to heat lysate for breaking protein-protein interaction and then do IP with this lysate. But I tried with somes condition such as 95*C in 5 min or 75*C in 15min but all samples got protein precipitation. So what is the correct heating condition? Please help me, thank you so much.
P/s: Lysis buffer is NP-40 lysis buffer
Did you try to use detergents for enhancing the solubility of the proteins? Use effective detergents, such as SDS, in your lysis buffer for enhancing the solubility of your proteins during heating.
Heating proteins causes denaturation (unfolding), which is why you used it to break protein-protein interactions. Unfortunately, denaturing proteins exposes their hydrophobic interiors, causing aggregation, which often leads to precipitation. The concentration of NP-40 you used was apparently insufficient to prevent this aggregation. You may be able to solve the problem by increasing the NP-40 concentration, but it might require a detergent with stronger solubilization properties, such as SDS, as suggested by Alemu Tekewe.
Another approach to denaturation is to use a chaotropic agent such as urea. This requires molar concentrations. The trick when using denaturing detergents like SDS or chaotropes like urea is to find a concentration that will destroy the protein-protein interaction you are interested in studying without preventing immunoprecipitation. After all, immunoglobulins are also proteins, and immunoprecipitation is also a protein-protein interaction. Fortunately, immunoglobulins are stabilized by several disulfide bonds, so it may be possible to find the necessary set of conditions, as long as you don't have a reducing agent in the buffer.
Another thought: If the protein-protein interaction you are studying involves electrostatic (charge-charge) interactions, you might be able to break it by adding a relative high concentration of salt.
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