Co-immunoprecipitation and semi-quantitative immunoblotting for the analysis of protein-protein interactions.
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ABSTRACT:
SUBMITTER: Burckhardt CJ
PROVIDER: S-EPMC8264609 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:MotivationAdvanced technologies are producing large-scale protein-protein interaction data at an ever increasing pace. A fundamental challenge in analyzing these data is the inference of protein machineries. Previous methods for detecting protein complexes have been mainly based on analyzing binary protein-protein interaction data, ignoring the more involved co-complex relations obtained from co-immunoprecipitation experiments.ResultsHere, we devise a novel framework for protein complex detection from co-immunoprecipitation data. The framework aims at identifying sets of preys that significantly co-associate with the same set of baits. In application to an array of datasets from yeast, our method identifies thousands of protein complexes. Comparing these complexes to manually curated ones, we show that our method attains very high specificity and sensitivity levels (? 80%), outperforming current approaches for protein complex inference.AvailabilitySupplementary information and the program are available at http://www.cs.tau.ac.il/~roded/CODEC/main.html.
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Project description:RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our results support a newly identified partially double-stranded UUUUUGAGA motif similar to that known for the splicing factor HNRNPC.
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Project description:Co-immunoprecipitation methods are frequently used to study protein-protein interactions. Confirmation of hypothesized protein-protein interactions or identification of new ones can provide invaluable information about the function of a protein of interest. Some of the traditional methods for extract preparation frequently require labor-intensive and time-consuming techniques. Here, a modified extract preparation protocol using a bead mill homogenizer and metal beads is described as a rapid alternative to traditional protein preparation methods. This extract preparation method is compatible with downstream co-immunoprecipitation studies. As an example, the method was used to successfully co-immunoprecipitate C. elegans microRNA Argonaute ALG-1 and two known ALG-1 interactors: AIN-1, and HRPK-1. This protocol includes descriptions of animal sample collection, extract preparation, extract clarification, and protein immunoprecipitation. The described protocol can be adapted to test for interactions between any two or more endogenous, endogenously tagged, or overexpressed C. elegans proteins in a variety of genetic backgrounds.
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