How to Choose the Appropriate Antibody in a Protein Testing Experiment
There are more than one kinds of antibody for selection to test target protein with any experimental purpose and even several kinds of antibody are included in one biological company. So it is crucial to choose an appropriate antibody in various kinds of antibodies. There are some elements for the antibody selection that you need to consider. The following tips are based on experimental methods, the species of experimental subjects, protein structure of specimen and species election of the antibody’s host.
First, the experimental method need to be determined through analysis.
Generally, the information that the antibody are available to some certain types of experiments listed in instruction book. For example, some antibody are applied to analysis of WB, IHC, ICC, but some are available to analysis of ELISA and FCAM. The unmentioned types of antibody dose not mean that this antibody fails to apply to the experiment and it means that the antibody has not been tested by this kinds of experiment When faced with this situation, the advice is to ask the seller for sample to determine whether to buy after testing. In addition, it would be listed in instruction book if this antibody fail to match a certain kinds of experiment.
Second, the species of experimental specimens have to be clarified.
What kinds of animal or human beings does the experimental sample belong to? It also plays an important role in the protein testing experiment. Normally, these information can be found in instruction book. For example, some antibodies are suitable for specimens of rat, mouse, human, pig and goat. Thus the same antibodies of same species or the antibodies containing cross reaction will be a good choice. Antibody might have cross reaction with same target protein of different species for the higher amino acid sequences. If the antibody’s species do not belong the listed catalogue of instruction book, it means that the species of experimental specimens has not been tested by the antibody instead of its failing to use. Therefore, try to avoid using this unmentioned antibody.
Third, analysis of protein structure of experimental samples will will be helpful to antibody election.
Two factors ought to take in to consideration. The first factor is domain of protein samples which is going to test. The antibody is produced by host with different immunogens and the immunogens contain full capsid protein, protein fragment, polypeptide,bacteria or cells. If it is the protein fragment or a part of full capsid protein that needs to be tested, it has to choose the antibody produced by the immunigens of the same part.
In addition, some certain antibodies requires special handing of experimental specimens in specimen collection. For example, many antibodies just have reaction with reduced, denatured and epitope-exposed protein samples, but some antibody recognized natural protein. When choosing immunohistochemical antibodies, it should consider that some antibody recognize some unfixed, frozen parts but some identify the parts with no antigen retrieval.
Fourth, selection of the host of antibody plays a vital role in the experiment.
Generally, when using coupling second antibody combining with primary antibody of no coupling., the selection of primary antibody’s host species own more significance. However, for immunohistochmistry, it recommend that choose primary antibody whose species are different from experimental sample’s to avoid second antibody have cross reaction with endogenous immune globulin of the sample.