I am currently working on optimizing a protocol for determining if two proteins are interacting in mammalian cells using pull downs. The protein of interest being over expressed in the system will be tagged with a 6xHis tag and then the cells will be infected with influenza. I want to pull down the 6xHis tagged protein using the Invitrogen dynabeads designed for 6xHis proteins. The only thing I can't find a clear answer to is whether freezing the samples after lysis in the manufacturer recommended non-denaturing lysis buffer (50mM NaPO4, 300mM NaCl, 1% Triton X-100) might affect the protein-protein interaction, or is it better to just go ahead and do the pull-down procedure immediately after lysing the cells and determining protein concentration? I appreciate any help the community can provide!
I don't think freezing will interfere with the protein-protein interaction, as long as it is done properly. The sample should be snap-frozen by immersion of the container in liquid nitrogen or dry ice. This prevents separation of solutes from the water in the sample. It would also be advisable to include a protease inhibitor cocktail in the lysis buffer. Be careful about the source of the Triton X-100. Old Triton can become chemically reactive and damage your sample.
I would recommend to add some cryoprotectant to prevent protein degradation whitch could lead to protein inability to interact. We use glycerol in final concentration of 20-25%. Be aware that it decreases the concentration of total protein.
- Badges
- Science topic
- Similar topics
- Filing