The RNAi Machinery Antibody Sampler Kit provides an economical means to analyze proteins associated with endogenous RNA interference. The kit contains enough primary and secondary antibodies to perform two western blot experiments.
Specificity / Sensitivity
Each antibody in the RNAi Machinery Antibody Sampler Kit recognizes endogenous levels of their target protein and does not cross-react with other proteins.
Source / Purification
Polyclonal antibodies are produced by immunizing animals with synthetic peptides corresponding to the residues surrounding Ala1123 of human Dicer or residues surrounding Leu118 of mouse Mili protein. Polyclonal antibodies are purified by protein A and peptide affinity chromatography. Monoclonal antibody is produced by immunizing animals with synthetic peptides corresponding to the sequence of mouse Argonaute 2 or around Gly953 of human Drosha proteins.
RNA interference (RNAi) serves as a global mechanism of gene regulation in eukaryotes. Through interactions with Dicer, Drosha, Argonaute 2 (Ago2) and Miwi/Mili proteins, microRNA (miRNA) is processed within the nucleus and utilized for gene silencing and down regulation of gene expression. Dicer is a member of the RNase III family that specifically cleaves double-stranded RNA to generate microRNA (miRNA) (1). Long, primary transcripts (pri-miRNAs) are processed to stem-looped pre-miRNAs by the nuclear RNase III Drosha (2) and are then transported to the cytoplasm for further processing by Dicer to produce mature, 22-nucleotide miRNAs (3). The mature miRNA then becomes a part of the RNA-Induced Silencing Complex (RISC) and can bind to the 3' UTR of the target mRNA (3). Interference of Drosha pri-miRNA processing results in the increase of pri-miRNAs and the decrease of pre-miRNAs (2). Drosha forms part of a multiprotein complex called the Microprocessor along with other components, such as DGCR8 (4). Both Drosha and DGCR8 are necessary for miRNA biogenesis (5). Argonaute protein family members participate in various steps of miRNA-mediated gene silencing such as repression of translation and mRNA turnover (6). The Drosophila piwi gene was identified as being required for the self-renewal of germ-line stem cells, and its homologues are well conserved among various species including Arabidopsis, C. elegans and human (7). Miwi and Mili proteins are both mouse homologs of Piwi and contain a carboxy-terminal Piwi domain that binds to Piwi-interacting RNAs (piRNAs) in male germ cells and are essential for spermatogenesis in mouse (8-11).