Biochemistry


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0 Q&A 6759 Views Feb 20, 2020
Protein-protein interactions constitute the molecular foundations of virtually all biological processes. Co-immunoprecipitation (CoIP) experiments are probably the most widely used method to probe both heterotypic and homotypic protein-protein interactions. Recent advances in super-resolution microscopy have revealed that several nuclear proteins such as transcription factors are spatially distributed into local high-concentration clusters in mammalian cells, suggesting that many nuclear proteins self-interact. These observations have further underscored the need for orthogonal biochemical approaches for testing if self-association occurs, and if so, what the mechanisms are. Here, we describe a CoIP protocol specifically optimized to test self-association of endogenously tagged nuclear proteins (self-CoIP), and to evaluate the role of nucleic acids in such self-interaction. This protocol has proven reliable and robust in our hands, and it can be used to test both homotypic and heterotypic (CoIP) protein-protein interactions.
0 Q&A 4535 Views Nov 20, 2018
In this protocol we describe the separation and visualization of ubiquitylated forms of the yeast mitofusin Fzo1 by Western blot. To this aim, we express HA-tagged Fzo1 in Saccharomyces cerevisiae, break the cells to extract a membrane-enriched fraction, solubilize the membranes using detergent and then specifically immunoprecipitate the tagged protein using anti-HA affinity beads. Subsequently, we separate the higher molecular weight (ubiquitylated) forms of Fzo1 via SDS-PAGE. Finally, immunoblotting and immunodecoration are used to detect the protein and its ubiquitylated forms using an HA-specific antibody. By using this protocol, it is possible to separate and visualize higher molecular weight forms of low abundant proteins such as Fzo1 and detect sharp and distinct bands above the unmodified protein by Western blot.
0 Q&A 9773 Views Jan 5, 2018
This protocol analyzes the direct interaction between two DNA-binding proteins by pull-down co-immunoprecipitation. One of the proteins is overexpressed in E. coli as HA-tagged recombinant protein and cell-free extracts are immunoprecipitated in HA-affinity resin. Cell extracts are treated with nuclease to degrade DNA and RNA, which rules out nucleic acid-mediated indirect interaction. Then, a second immunoprecipitation step is performed using the purified putative partner protein. Co-immunoprecipitated proteins can be detected either by Coomassie Blue staining and/or Western blotting (WB) if a specific antibody is available. Moreover, many DNA/RNA binding proteins are highly electropositive, which can hinder WB under standard conditions, as has been shown in histones and histone-like proteins. In this case, we show that the high isoelectric point of the putative partner results in a poor transfer. Tips to troubleshot WB transfer of highly electropositive DNA-binding proteins are provided.
0 Q&A 10474 Views Aug 20, 2017
Pulse-chase technique is a method widely used to assess protein or mRNA stability. The principle of pulse-chase relies on labeling proteins or mRNA produced during a short period of time called ‘pulse’ and then following the rate of disappearance of those labeled proteins over a period of time called ‘chase’. This technique thus allows quantitative analysis of modulation of protein or mRNA stability under different treatments or culturing conditions.
0 Q&A 21125 Views Jul 5, 2015
Nicotiana benthamiana (N. benthamiana) is a useful model system to transiently express protein at high level. This protocol describes in detail how to transiently express protein in N. benthamiana and how to carry out protein immunoprecipitation in this expression system. This protocol can be broadly used for investigation on protein-protein interaction, protein purification and other related protein assay.
0 Q&A 13933 Views May 20, 2015
Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014).
1 Q&A 17219 Views Apr 5, 2015
Immunoprecipitation (IP) is a biochemical technique to precipitate a protein out of solution using an antigen that can specifically bind to that protein. IP can be performed to isolate and concentrate one particular protein from a sample of thousands of different proteins. IP is also readily performed to pull down interacting proteins of complexes out of solution. This protocol outlines the methods used to IP proteins in whole worm lysates and their preparation for detection on Western blots using denaturing conditions.
0 Q&A 13942 Views Apr 5, 2014
The fate of mRNA, in particular its stability, localization and rate of translation is regulated by RNA binding proteins assembling to messenger ribonucleoprotein (mRNP) complexes. To investigate the transcriptome-wide RNA binding sites of UPF1, the core factor of nonsense-mediated mRNA decay (NMD), we performed individual-nucleotide-resolution UV cross-linking and immunoprecipitation (iCLIP) (Zund et al., 2013) followed by high-throughput sequencing. The presented protocol is optimized to investigate the RNA-binding sites of UPF1 and is based on previously described studies (Konig et al., 2010; Konig et al., 2011; Hafner et al., 2010). We want to thank the Group of Mihaela Zavolan (Swiss Institute of Bioinformatics, Basel, Switzerland) and Jernej Ule (Medical Research Council Laboratory of Molecualar Biology, Cambridge, UK) for technical support in setting up these experiments.
0 Q&A 10511 Views Apr 5, 2014
UPF1, an RNA helicase and a core factor of nonsense-mediated mRNA decay (NMD), interacts with RNA independently of the sequence context. To investigate the influence of translation on the association of UPF1 with specific reporter transcripts, UPF1 RNA immunoprecipitations (RIPs) are performed from Hela cells that either express a normally translated immunoglobulin-µ (Ig-µ) reporter (mini µ) or a version with a stable stem loop in the 5' UTR (SL mini µ) that efficiently inhibit translation initiation (Zund et al., 2013). Both the cloning of the SL mini µ reporter construct and the UPF1 RIP experiment are described in detail.
0 Q&A 9585 Views Mar 5, 2014
Co-immunoprecipitation assay of TLR3-Flag or Myc-MSR1 with HCV RNA is used to identify direct interaction of viral RNA with host proteins that recognize viral RNA to initiate interferon (IFN) signaling, a crucial antiviral response of the host cells. Both Toll-like receptor 3 (TLR3) and class-A scavenger receptor type 1 (MSR1) proteins recognize viral double-stranded RNA (dsRNA) which may be released into the extracellular milieu or spread from HCV-infected cells to uninfected neighbor cells via cell-to-cell contact, resulting in IFN-β activation that restricts viral propagation. We have found that MSR1 binds extracellular dsRNA, mediating its endocytosis and transport toward the endosome where it is engaged by TLR3, thereby triggering IFN responses in both infected and uninfected cells. We used this assay to demonstrate the pivotal role of MSR1 in mediating TLR3-recognition of the HCV RNA. The assay described in this protocol is based on the conventional protein immunoprecipitation protocol with conditioned buffers that prevent nonspecific RNA degradation by RNase present in the lysate. RNA molecules associated with the Flag-tagged protein were trapped by a specific antibody followed by Protein G capture, extracted and detected quantitatively by RT-PCR assay, followed by agarose-gel electrophoresis for visualization. This method can also be applied to detection of other protein-RNA interactions.



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