The immunoprecipitation is an immunoassay often used which uses immobilized antibodies on a solid support to isolate, from a complex sample, a specific protein.
The applications of immunoprecipitation range from the study of the presence or relative abundance of a certain protein, to studies of functionality and interaction between proteins and studies of post-translational modifications or expression profiles.
In this post we bring you some keys and critical aspects for a successful immunoprecipitation assay .
1.- SAMPLE PREPARATION
Before carrying out the immunoprecipitation protocol, it is necessary to extract the cellular antigens , making the lysate buffer a critically important element in obtaining a quality sample. Ideally, this buffer should stabilize the native conformation of the protein, inhibit enzyme activity, avoid denaturation of the antibody binding site, and ensure maximum antigen release from the starting cells or tissues.
To avoid proteolysis, denaturation and dephosphorylation phenomena, once the antigen has been extracted from the original sample, it must be kept cold. We will also reduce the risk of these phenomena by adding phosphatase and protease inhibitors to the lysate buffer.
Subsequently, the extracted antigen must be pre-clarified by purification with protein A or G to eliminate other unwanted antigens that may be included in the sample, and which could lead to non-specific binding.
2.- SELECTION OF ANTIBODIES
As a general rule for immunoprecipitation assays, a polyclonal antibody will be chosen as the capture antibody. The main reason is that polyclonal antibodies bind to multiple epitopes of the same target protein, and form stronger immunocomplexes than monoclonal antibodies.
You can expand the information on the pros and cons of polyclonal and monoclonal antibodies in this entry .
3.- ANTIBODY PURIFICATION METHOD
The polyclonal antibodies used in immunoprecipitation assays can be in the form of an antiserum, precipitated with ammonium sulfate, or affinity purified with protein A or G. The latter are preferred since they have a higher degree of purity and are more specific and therefore they will reduce non-specific junctions and background noise.
4.- ANTIBODY TITLE
Antibody titration is a fundamental step to optimize the result of our immunoprecipitation assay. In this way we will avoid that the immunoassay turns out to be inefficient (due to lack of antibody) or non-specific (due to excess of antibody).
As always, for the correct interpretation of the results, also in the immunoprecipitation assays it is essential to include a series of controls, both positive and negative.
6.- WASHING STEPS
The washing steps are critical to obtain a specific result. With them we will eliminate proteins that have not bound to the antibody, thus avoiding nonspecific signals.
It is necessary to optimize the washing steps, since an excessive washing could lead to a reduction of the antigen-antibody binding.
In addition to the lysate buffer that we have discussed in point 1, the challenge of buffers to be used in the immunoprecipitation assay such as the binding buffer, the wash buffer and the elution buffer must be properly selected.
- Binding buffer
Generally, antigen-antibody interactions occur in practically any buffer with a pH close to neutral, such as PBS or TBS, although in certain cases the use of more specific binding buffers will have to be evaluated.
- Wash buffer
It should favor conditions in which the interaction with the protein of interest is maintained, but the binding of non-specific proteins is avoided. Generally, buffers with saline concentrations and physiological pH such as PBS or TBS are used, and the reagents that are necessary in each case are added.
- Elution buffer
The elution buffer must have the necessary strength and pH to ensure the correct elution of the proteins from the beads .