In my co-immunoprecipitation research, how can I set up a control?
Controls are essential to ensuring the specificity and validity of your co-immunoprecipitation experiment. Several forms of regulation are listed below.
1. IgG controls are negative controls; you should add an IgG control for non-specific binding. An IgG secondary antibody should not recognize any of the components in your lysate but should be of the same isotype and species as your primary antibody.
Controlled Absence of Antibodies: To test for non-specific protein binding to the beads or non-specific precipitation, continue with the co-immunoprecipitation technique as usual, but leave out the primary antibody.
2. Use a set of proteins already known to interact as a positive control, if at all feasible. In this way, the efficiency of your co-IP protocol may be confirmed.
Load regulation (or input regulation): A fraction (typically 5–10 percent) of the cell lysate from your immunoprecipitation studies is put onto the gel to indicate that the target protein was present in the original sample.
3. Thirdly, pre-clearing lysates are used as test controls. Agarose bead binding is a step that is done before co-IP to get rid of any proteins in the samples that do not specifically bind to the beads. The noise level in co-IP studies may be decreased with the use of this parameter.
The immunoprecipitated protein western blot test is done to make sure that the target protein was successfully immunoprecipitated after the co-IP proteins have been separated on a gel.
Consider the specifics of your experimental design and goals when deciding which controls to use. The highest level of scientific rigor and confidence in your findings depends on including these crucial controls.