Immunoprecipitation

coming soon, please be patient

 

In the meanwhile, there are some interesting papers which could show the Ago2 load of siRNA or antagomir therapeutics in vivo, certainly by applying Ago2 immunoprecipitation. At the same time this sort of ligand binding assay (with Ago2 being the ligand) is the most sensitive one if isolating microRNA or siRNA drugs from biological samples. Recently the Ago2 immunoprecipitation assay was used to identify numerous small RNA populations like piRNA (piwi-interacting RNA).

Argonaute 1 (Ago1) is also a cytosolic RNA dependent endonuclease which is guided to the target mRNA by microRNAs (miRNA), at least in Drosophila (5).

Immunoprecipitation was is an interesting tool to study RNA binding to Toll-like receptors or the retinoid-inducible gene 1 (RIG1). Monoclonal antibodies specific for TLR3, TLR7, and TLR8 were used to immunoprecipitate messenger RNA therapeutics bound to this proteins of the innate immune system (6).

There are monoclonal antibodies detecting 5-methylcytidine (m5C) modifications of genomic DNA. The immunoprecipitation of methylated genomic DNA for pre-transcriptional gene silencing studies is greatly enhanced by this specific antibody.

Immunoprecipitation is also a widely used method to remove antigen/antibody complexes from the liquid phase in radioimmunoassays (7). Briefly, labelled antigen competes with unlabelled antigen bound to antibodies in the sample. The more unlabelled antigen is present, the more labelled antigen is displaced from the antigen/antibody complex. After immunoprecipitation with anti-IgG antibodies, the radioactivity in the pellet is measured.

 

References:
(1) Pei Y et al (2010), RNA 16: 2553-2563. (the fist Merck paper on this topic)
(2) Wei J et al (2911), Mol Pharmacol doi:10.1124/mol.110.070409 (also Merck scientists)
(3) Obad S et al (2011), Nature Gen 43(4): 371-8. (Santaris┬┤ tiny LNA antagomirs).
(4) Hess AM et al (2011), BMC Microbiol 11: 45
(5) Ameres SL et al (2010), Science 328: 1534-9.
(6) Kormann MSD et al (2011), Nature Biotech 29(2): 154-7.
(7) Schramm W et al (1987), Clin Chem 33(8): 1331-7.

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