In Western Blotting (WB), in an ideal world, an antibody only binds to the protein of interest. Therefore only one band should be visible on your gel and the thickness of the band should correspond to the amount of protein that is present. This is not always the case and in many instances the antibody catches additional bands above and below the protein of interest. When this happens quantification is compromised since these non-specific proteins overshadow the correct bands in intensity. Antibodies have a spectrum of affinity to a wide variety of proteins. The choice of primary antibody for the experiment depends on the antigen you want to detect and on how the protein of interest folds, as it exposes different epitopes under different conditions. Antibody selection and calibration are important first steps. Both polyclonal and monoclonal antibodies, labeled or unlabeled, can work well.
Selecting a primary antibody is only a first step, it is critical to optimize its concentration by running a calibration curve. The importance of using a well validated antibody and inclusion of proper experimental controls is essential to the success of your experiment. We all know that there is a wide selection of primary antibodies on the market, unfortunately the quality can differ greatly. It is the responsibility of the scientist to properly test antibodies and all reagents before proceeding with any WB experiment. The primary antibody should be thoroughly tested, validated to be specific, sensitive, and reproducible enough to detect the target protein of interest when used at the lowest concentration. The secondary antibody used in WB can also contribute to non-specific binding to various proteins which can lead to high background. It is recommended to also test the quality of the secondary antibody; this can be done by performing a blot without using primary antibody followed by film exposure. Due to the cost of primary antibodies performing a titration experiment to identify the best working concentration can reduce cost and confer better results. Precision Biosystems BlotCycler–– touch has the ability to automate the titration process; up to six different concentrations of primary antibody can be run simultaneously. After chemiluminescence detection, the six blots can be compared to determine the best antibody concentration for the target protein.
