When designing IHC/ICC experiments, the choice of primary antibody is very important. All steps of the IHC/ICC experiment must be optimized in order to observe specific staining and minimize non-specific background staining. This includes conducting preliminary experiments to determine the appropriate incubation conditions for each primary antibody. In order to obtain reliable and specific signals, high-quality antibodies with minimal cross-reactivity should be used.
Generally, the antibody instructions list the type of analysis that the antibody has been verified to be suitable for. If the application scope is not mentioned in the instructions, it does not mean that the antibody is not suitable for this application. It just means that it has not been verified by this kind of analysis and experiment, and you need to explore and optimize the conditions by yourself.
Understanding the structural properties of the target protein helps to choose the most suitable antibody. At least two factors need to be considered, the domain of the target protein to be tested and the extraction or processing process of the sample.
Should choose antibodies of the same species. Antibodies may cross-react with the same target protein of different species because of the high amino acid sequence homology. The method of sequence alignment should be used to predict the cross-reactivity. Expasy and NCBI BLAST can be used to compare the homology of proteins of different species.
When using a conjugated secondary antibody combined with a primary antibody without a conjugate, the species selection of the host animal of the primary antibody is more important. For immunohistochemistry, try to choose the primary antibody of a species different from the sample to avoid cross-reaction between the secondary antibody and the endogenous immunoglobulin of the sample.
Polyclonal antibodies are heterogeneous and can recognize multiple epitopes, so they are less affected by changes in protein conformation. Generally speaking, polyclonal antibodies are more stable than monoclonal antibodies within a certain range of pH and salt concentration. For these reasons, polyclonal antibodies are more commonly used in IHC/ICC experiments.
The concentration of primary antibody, dilution, incubation time and temperature all affect the quality of staining. These variables need to be optimized for each antibody and sample in order to achieve specific staining and low background. The optimal antibody concentration that can achieve the best staining effect and the lowest background signal must be individually determined experimentally in each analysis. The commonly used method is a titration experiment involving a series of dilutions. When performing a titration experiment, a fixed incubation time should be selected first, and then a series of experimental dilutions of antibodies should be prepared. Each dilution should be tested with the same type of sample to maintain the same experimental conditions.
Creative Bioarray's experienced scientists team offer customized antibody selection and optimization services for IHC, ICC and IF applications depending on your research needs. Optimization will be performed using different pre-treatment methods and dilutions of antibody.
Please contact us for more details.