What Are the Different Types of Immunoprecipitation?
Immunoprecipitation (IP) refers to the method of separating proteins in a solution using a specific antibody coupled to a solid substrate (e.g., agarose resin or magnetic particles). After the target protein is isolated, subsequent detection and analysis can be performed through western blotting and other assay techniques to determine its identity, structure, expression, and modification state.
Types of Immunoprecipitation
There are several variations of immunoprecipitation used in the lab. These include individual protein immunoprecipitation (IP), co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), ribonucleoprotein (RNP) immunoprecipitation which includes both RNA immunoprecipitation (RIP) and crosslink immunoprecipitation (CLIP), and tagged protein-IP.
Individual protein immunoprecipitation (IP)
By far, this is the simplest form of IP in the lot. In this method, a single protein is isolated from a complex crude lysate solution (plant or animal tissue and other biological samples) using an antibody that specifically binds to that particular protein.
Co-immunoprecipitation (Co-IP)
This technique generally follows the same principles as IP, but instead of isolating individual proteins, it precipitates intact protein complexes (i.e., the target antigen and its binding partners) out of the solution. In co-IP, an antibody with a strong affinity to one of the identified components of the protein complex is used to isolate or “pull down” the entire protein complex out of the solution.
Co-IP is routinely used in laboratories for investigating and analyzing protein-protein interactions.
Chromatin immunoprecipitation (ChIP)
Chromatin immunoprecipitation is commonly used in identifying the location of DNA binding sites on the genome of the target protein. Thus, it provides a more in-depth understanding of the protein-DNA interactions occurring inside living cells.
In this method, the DNA and the DNA-binding proteins are crosslinked using formaldehyde or other crosslinkers such as DTBP. After crosslinking, the cells are lysed and the DNA undergoes sonification to break it into pieces. At this point, the protein-DNA complex is immunoprecipitated out of the cellular or tissue lysate using an antibody specific to the putative DNA-binding protein.
Finally, the DNA is released by heating the purified DNA-protein complexes (which break the formaldehyde crosslinks) and the identity and quantity of the DNA of interest is determined through polymerase chain reaction (PCR).
RNP precipitation (RIP and CLIP)
Both of these methods are extremely useful in identifying bound RNAs and play a huge role in understanding ribonucleoproteins.
In RNA immunoprecipitation chip (RIP), the RNA-binding proteins are immunoprecipitated out of the sample solution and identified through RT-PCR and complementary DNA (cDNA) sequencing. While this technique is generally used to determine the RNA sequences interacting with a particular RNA-binding protein in vivo, it can also be used to establish relative levels of gene expression, identify RNAs with related functions, and detect subsets of RNA which may be co-regulated.
On the other hand, crosslink immunoprecipitation (CLIP) elucidates the mechanisms and functions of the post-transcriptional regulatory networks by identifying the RNA binding sites of proteins. In this method, the cells are UV crosslinked in vivo prior to lysis and the RNA is subjected to partial fragmentation before isolating the target protein via immunoprecipitation. The protein of interest is then removed from the crosslinked RNA by performing proteinase K digestion and cDNA is synthesized through RT-PCR.
Tagged-protein IP
Generally, the IP methods discussed earlier are largely dependent on the availability of antibodies that specifically targets the protein of interest. To solve this problem, proteins can be tagged with short peptide sequences or fluorescent proteins to which a high-affinity antibody is available.
Also known as tagged-based pull-down, tagged-protein IP is now used for all types of immunoprecipitation techniques. Some of the most popularly used tags in IP include glutathione-S transferase (GST), green fluorescent protein (GFP), and FLAG peptide. Likewise, researchers also use the c-Myc tag (peptide sequence EQKLISEEDL), hemagglutinin (peptide sequence YPYDVPDYA) tag, and V5 tag (peptide sequence GKPIPNPLLGLDST) in tagged-protein IP.
Image: HOOK™ GST Protein Purification
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