Immunoprecipitation (IP) is a technique to isolate a specific protein out of a solution using an antibody that binds to it. Antibody-protein complexes are removed from the solution with the addition of an insoluble form of an antibody binding protein, such as Protein A or Protein G conjugated to agarose slurry or the newly popular Magnetic Beads. Immunoprecipitation assays detect the interaction of a target protein with other proteins or nucleic acids. Assay examples include Co-IP, ChIP, RIP and tagged protein IP.
Co-Immunoprecipitation (Co-IP) is a powerful method that is most widely used by researchers to analyze protein–protein interactions. This process provides a rapid and simple method to separate a specific protein from a sample containing thousands of different proteins, such as serum, cell lysate, homogenized tissue or conditioned media.
Chromatin Immunoprecipitation (ChIP) is a type of immunoprecipitation used to investigate regions of genome associated with a target DNA-binding protein, or conversely to identify specific proteins associated with a particular region of the genome. The primary applications of ChIP are as follows:
RNA Immunoprecipitation (RIP) is performed with an antibody that targets a specific RNA-binding protein. By isolating the protein, the RNA bound to it is also isolated. The RNA-protein complexes are separated by RNA extraction. The RNA can be analyzed by cDNA sequencing or RT-PCR.
Traditional immunoprecipitation (IP) uses agarose beads coated in protein A/G. Most of these IP protocols require more than three washing steps to eliminate background and non-specific binding. Each step of washing causes some bead loss or leaves behind residual contaminants.
The use of magnetic beads has recently gained popularity as a quicker, more accurate approach for immunoprecipitation. Unlike agarose, no columns or centrifugations are required. Also, you can significantly reduce experiment variability by being able to remove all supernatant without disturbing your pellet. You will consistently get more reliable and reproducible data with GenScript MagBeads.
This protocol offers a general guideline for immunoprecipitation with GenScript MagBeads.
A. Cross-linking IgG to the Beads
B. Binding Target Protein to the IgG Cross-linked Beads
C. Elution of Target Protein