It is known that immunoprecipitation is a method to test target protein or antigen in solution. This method has been used in the research of protein, gene and other biological areas. It has a important place in biological experiments. Every method to immunoprecipitation has its certain operation process, but most of them are based on the specificity between antibody and antigen. The normal way to use the covalently bound Protein A , the agarose or magnetic microspheres of protein G to purify the compound of antigens and antibody. Thus protein A and protein G can bind with the conserved region of antibodies to form a steady compound of antigens and antibodies which is attached to magnetic microspheres. When the unrelated substance in solution is removed by washing magnetic microspheres, antigens or molecules bound with antigens can be purified. When do this experiment, some important factors need to be taken into account.
First factor is specimen. The key of the success of immunoprecipitation is primary sample preparation. Immunoprecipitation experiment aims to test the interaction between antibody and antigen, so the quality of sample determines the quality of the interaction between antigens and antibodies. Preparing samples needs to choose an appropriate way according to the resource of antibody samples including tissue samples and cells. Then place the antigens which is to be tested in sample solution to release. It requires adding proper amount of protease into lysis buffer to avoid degradation of antigens and compounds. In addition, it is suggested to choose the lysis buffer containing proper disincrustant to make sure that these cells can release antigens.
The second significant factor is antibody. When choose antibody, not only the specificity but also affinity of antibodies have to be taken into account. Polyclonal antibody only has a good specificity to single epitope of antigen and it may cause that epitope of antigen can not get enough exposure and get damaged. Thus antibody cannot recognize antigen, which stocks producing immune complex. It will have a bad effect in the result of immunoprecipitation. However, monoclonal antibody or mixed polyclonal antibody can bind with several epitopes of antigen. They can solve the problem of affinity. But not all kinds of antibodies can be applied to immunoprecipitation, so pay more attention to
choosing antibody.
Third factor is magnetic microspheres. The microspheres which are used widely in immunoprecipitation is agarose and magnetic microspheres. They all have its unique features. But the latter becomes rising star, because the heart is superparamagnetism grain and the it is wrapped by a smooth substance. It can binds with more micro proteins and separate the target proteins. So magnetic microspheres can save much time and it earns good reputation.
Application