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Protein-protein interactions play key roles in nuclear processes including transcription, replication, DNA damage repair, and recombination. Co-immunoprecipitation (Co-IP) followed by western blot or mass spectrometry is an invaluable approach to identify protein-protein interactions. One of the challenges in the Co-IP of a protein localized to nucleus is the extraction of nuclear proteins from sub-nuclear fractions without losing physiologically relevant protein interactions. Here we describe a protocol for native Co-IP, which was originally used to successfully identify previously known as well novel topoisomerase 1 (TOP1) interacting proteins. In this protocol, we first extracted nuclear proteins by sequentially increasing detergent and salt concentrations, the extracted fractions were then diluted, pooled, and used for Co-IP. This protocol can be used to identify protein-interactome of other chromatin-associated proteins in a variety of mammalian cells.Microdissection techniques are very important for anatomical and functional studies focused on neuroscience, where it is often necessary microdissect specific brain areas to perform molecular or anatomical analyses. The parafilm®-assisted microdissection (PAM) was previously described and involves the microdissection of tissue sections mounted on parafilm-covered glass slides. In this work, we describe the use of the PAM method to microdissect rodent nucleus accumbens (NAc). (1) We first describe the best way to perform the mouse euthanasia and how to remove the brain. (2) Next, we describe how to prepare the slides with parafilm® that will be used to receive the brain slices. (3) Following, we describe how to handle the brain in the cryostat, how to align the hemispheres and how to identify the NAc antero-posterior limits. (4) We also describe how to perform the staining and dehydration of the slices, a critical step to facilitate the microdissection and preserve macromolecules. click here (5) In the final step, we describe how to identify the dorso-ventral and latero-medial limits of the NAc and, finally, how to perform the manual microdissection of the area. This is a low-cost technique that allows the researcher to specifically microdissect any brain region, from which intact RNA and proteins can be extracted to perform several molecular analyses (e.g., real-time PCR, Western blot, and RNA-seq).Ribosomes are an integral part of cellular life. They are complex molecular machines consisting of multiple ribosomal proteins and RNAs. To study different aspects of ribosome composition, many methods have been developed over the decades. Here, we describe how to purify ribosomes from the α-proteobacterium Rhodopseudomonas palustris. Following this protocol, RNA can be extracted from either purified ribosomes or directly from cell cultures, and ribosomal RNAs quantified using Northern blot. This protocol gives an example of studying ribosomes in a bacterium other than the commonly used E. coli. The challenge of performing Northern blots with rRNA is also addressed in detail.Transfer RNA (tRNA) is an essential link between the genetic code and proteins. During the process of translation, tRNA is charged with its cognate amino acid and delivers it to the ribosome, thus serving as a substrate of protein synthesis. To analyze the charging state of a particular tRNA, total RNA is purified and analyzed on an acid-urea gel. Separated RNA is then transferred to a membrane and detected with a probe for the tRNA of interest. Here, we present an improved protocol to analyze the tRNA charging state in the α-proteobacterium Rhodopseudomonas palustris. Compared to the classical method, the RNA isolation step is optimized to suit this organism. Additionally, a non-radioactive platform is used for electrophoresis and Northern blots. This significantly reduces the time and the effort required for this protocol.Chromatin Immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) or high-throughput sequencing (ChIP-seq) has become the gold standard for the identification of binding sites of DNA binding proteins and the localization of histone modification on a locus-specific or genome-wide scale, respectively. ChIP experiments can be divided into seven critical steps (A) sample collection, (B) crosslinking of proteins to DNA, (C) nuclear extraction, (D) chromatin isolation and fragmentation by sonication, (E) immunoprecipitation of histone marks by appropriate antibodies, (F) DNA recovery, and (G) identification of precipitated protein-associated DNA by qPCR or high-throughput sequencing. Here, we describe a time-efficient protocol that can be used for ChIP-qPCR experiments to study the localization of histone modifications in young inflorescences of the model plants Arabidopsis thaliana.Electron cryotomography (cryo-ET) is an increasingly popular technique to study cellular structures and macromolecules in situ. Due to poor penetration of electrons through thick biological samples, the vitreously frozen samples for cryo-ET need to be thin. For frozen-hydrated cells, such samples can be produced either by cryomicrotomy or cryo-FIB-milling. As a result, a tomogram of such a sample contains information of a small fraction of the entire cell volume, making it challenging to image rare structures in the cell or to determine the distribution of scattered structures. Here, we describe the tools and workflow that we designed to facilitate serial cryomicrotomy, which makes possible the exploration of a larger volume of individual cells at molecular resolution. We successfully used serial cryomicrotomy to locate and image the Dam1/DASH complex located at microtubule plus ends inside mitotic Saccharomyces cerevisiae cells.Sweet basil (Ocimum basilicum) is a popular herb with high economic value and is currently threatened by a severe oomycete disease. An efficient transformation method is a prerequisite for gene functional analysis to accelerate molecular breeding and deploy effective disease management strategies, and breeding through genetic engineering. Here we present a detailed protocol for a highly efficient Agrobacterium tumefaciens-mediated transformation method for sweet basil, which was established based on a previously reported method by other researchers, with modifications on several aspects, including growth of sweet basil, age of plants used for explants, preparation and concentration of Agrobacteria. This protocol allows researchers in academia and agroindustry to generate transgenic sweet basil plants in an easy, quick and highly reproducible manner. In addition, this protocol may be applicable to transform other species within the genus Ocimum.