IP sample treatment
IP Sample treatment:
The essence of the immunoprecipitation is the interaction of antibodies and antigens with natural conformation. The antigen’s state, concentration and conformation are largely affected by how samples are treated. Therefore researchers should pay attentions to how to make qualified samples. Typically, in this step, there are two tips: first, make sure samples are treated on ice or at 4°C; second, mind the ingredients of the lysis buffer.
The samples applied in IP are normally primary culture lysate or cell line lysate. Take RIPA lysis buffer as an example, here we list the functions of varied components, thus researchers can make their own lysis buffer recipes according to properties of target proteins and experimental purpose.
- Buffer: ionic buffer is often Hepes or Tris-HCl, pH=7.4
- The concentration of NaCl is normally 150mM. This concentration is close to physiological condition so interactions of proteins won’t be disrupted. Intracellular NaCl is unevenly distributed. In certain subcellularly locations NaCl can be 50mM. Therefore the optical concentration of NaCl should be determined by subcellular localization of target protein.
- Glycerol can protect the proteins’ interactions by its viscosity. Generally adding of 10% glycerol will help stabilize the proteins’ interactions.
- The detergent in lysis buffer lyses the cytoplasmic membrane. At the same time it destructs membranes of organelles and release proteins and proteinases in them. Activities of most proteinases are kept because the detergents used in IP are mild. Some other proteinases may be from the cytoplasm. Therefore, to prevent the degradation of the target protein, adding protease inhibitors is critical. Usually, EDTA is added to inhibit metalloproteinases, and protease Cocktail (a variety of protease inhibitor mixture) is added to inhibit proteinases.
- Different kinds and concentrations of detergents affects IP in 3 aspects:
Permeability of cytoplasm/organelles membranes:
Because most target proteins are localized in organelles, only when they are released can antibodies react with them.
Release of membrane proteins:
Conformations of many membrane proteins are sensitive to concentrations of detergent, thus to perform IP of these proteins, one needs to try different kinds and concentrations of detergents.
The extent to which protein interactions are effected differs by sorts of detergent. Since it’s hard to decide which kind of detergent is suitable for specific protein, it’s practical that researchers find a suitable detergent and optical concentration in pre-experiment.