Antibody refers to the immunoglobulin produced by plasma cells differentiated by B lymphocytes under the stimulation of antigens, which can specifically bind to the corresponding antigen. So in the experiment,Used for WB, IP, IHC, IF, CHIPofWhat are the differences between antibodies?
Western blot (WB) is a very common molecular biology experimental technique in the field of life sciences, which is based on the principle of using SDS-PAGE to separate proteins, then transfer the proteins to the membrane, and then use antibodies to detect proteins. During the experiment, the protein is linearized due to the thermal denaturation of the protein before electrophoresis, which breaks the hydrogen bonds that maintain the secondary and ** structure of the protein, and the antibody that recognizes the linear epitope is required. Therefore, WB antibodies are generally prepared by using synthetic peptides as antigens.
IHC (immunohistochemistry) is a technology that uses labeled antibodies to study antigens in cells or tissues through antigen-antibody specific immune response and chemofluorescence color reaction. In the process of immunohistochemistry experiments, tissues or cells need to be fixed to maintain the consistency of cell morphology and structure in their native state, and to ensure the stability of the antigen specificity of tissue cells. However, the immobilization of chemical substances will denature the protein, which will be different from the protein structure in its native state, and different from the structure of heating and denaturation in WB.
Therefore, in IHC IF experiments, the most suitable is the antibody prepared by using the recombinant protein as the antigen, and the antibody can also be prepared by the artificial synthesis of polypeptide as the antigen. If the antibody is being used to recognize a linear epitope (e.g., several amino acids in a row on a protein), then the antibody can be used for both IHC and WB, whereas if the antibody recognizes a conformational epitope, it can only be used for IHC.
IP (Immunoprecipitation): A method of purifying and enriching a protein of interest in a sample by a specific reaction of antigen and antibody. In general, the target protein and its corresponding antibody (Protein A G) are precipitated by forming an immune complex. IP technologies can be divided into the following three types according to the different detection targets: chip, RIP, and co-LP. Chip is the detection of the amount of DNA bound to proteins after enrichment using IP technologyRIP is the amount of RNA that is detected to which it binds;Co-IP is the amount of protein that is measured to which it binds.
Immunoprecipitation is to use antibodies to recognize and bind proteins in their natural state, so it is best to choose antibodies prepared from natural proteins as antigens when doing IP experiments, and of course, antibodies prepared from recombinant proteins as antigens can also be selected.
That's itUsed for WB, IP, IHC, IF, CHIPofWhat are the differences between antibodies?