scientificprotocols

Authors: Swagata Ghosh, Hanumantha Rao Kongara & Asis Datta 

Abstract

Considerable effort has been made in the past decade to unravel biological networks like protein-protein interaction. Various kinds of metabolite including small metabolites comprise a vast majority of cellular component. Hence a technique that identifies endogenous protein-metabolite interaction can reveal extensive roles of metabolites in regulation of protein activities. Several studies that feature on biological networks utilize Saccharomyces cerevisiae as the model organism. The pathogenic yeast Candida albicans remains understudied till date. We describe a new methodology that helps in identifying metabolites bound to proteins in vivo in Candida albicans. The technique employs yeast based Tandem Affinity Purification followed by methanol extraction and subsequent identification of the metabolite by UPLC- coupled ESI Mass Spectrometry.

Procedure

1. Suspend frozen yeast cell pellets from 500-ml cultures in 5 ml lysis so

scientificprotocols

Authors: Holger Kramer & Benjamin G. Davis 

Introduction

Methanethiosulfonates (MTS) are a group of reagents which allow the site-selective modification of accessible cysteine residues in proteins. (1) Synthesis of compounds carrying the MTS moiety has allowed the covalent attachment of structures to protein surfaces which represent functional mimics of post-translational modifications. (2)

Here we describe the synthesis of ethyl-linked O-glycosyl methanethiosulfates which allow the selective incorporation of fully deprotected carbohydrate structures onto protein scaffolds. (3,4)

scientificprotocols

Authors: Holger Kramer & Benjamin G. Davis 

Introduction

C-glycosides have received considerable attention in synthetic carbohydrate chemistry due to their unique features. As opposed to their natural congeners they are not prone to enzymatic or chemical hydrolysis. They can be prepared as anomerically pure glycosides and are isosteric to the heteroatom containing counterparts. (1)

Here we describe the synthesis of an alkynyl C-galactoside which was successfully employed in protein modification reactions by Cu(I) catalysed triazole formation. (2) The syntheses of the corresponding C-glucoside (3) and C-mannoside (4) have been described previously in the literature. (3,4)

scientificprotocols

Authors: Wei Shi

Abstract

We describe a powerful and easy-to-use RNA-seq analysis pipeline that can be used for complete analysis of RNA-seq data. It starts with raw read output of an sequencing instrument and reports lists of genes that are found to be differentially expressed in the comparison of different cell types. It consists of several analysis modules including Subread read alignment [1], featureCounts read summarization [2], voom normalization [3] and statistical testing of differential expression using empirical Bayes moderated t-statistic [4]. The entire pipeline mainly makes use of two R packages, Rsubread and limma, both available from the popular Bioconductor project.

Procedure

1. Check the sequencing quality of RNA-seq data using ‘qualityScores’ function in Rsubread package.

• Use ‘align’ function in Rsubread to align the reads.

scientificprotocols

Authors: Shan Sockanathan

Abstract

The detection of transcripts in sectioned tissue by in situ hybridization is a useful method to localize sites of gene expression during development. This protocol is one that we have modified from Scharen-Wiemers and Gerfin-Moser Histochemistry 100:431-440.

Introduction

This protocol describes a step by step method to detect mRNA transcripts on tissue sections cut using a cryostat.

scientificprotocols

Authors: Ronald Holewinski, Mark Ranek, David Kass & Jennifer Van Eyk 

Abstract

Protein phosphorylation is one of the most routinely studied post-translational modifications and is involved in a variety of cellular signaling processes. Currently, mass spectrometry is the analysis of choice for detecting global phosphoproteomic changes in biological systems, with the ability to map 100s-1000s of phosphorylation amino acid residues. In the current protocol we describe the enrichment and detection of phosphopeptides from cultured neonatal cardiac myocytes. This in vitro method allows for manipulations of the cellular environment that are impossible to obtain with in vivo models and have been a valued commodity in the unraveling of cardiac signaling pathways.

Introduction

The evolution of proteomics-based mass spectrometry has greatly expanded the understanding of protein post-translational modifications (PTMs) and has allowed for the un-biased analysis of a variety of PTMs and their potentia

scientificprotocols

Authors: Jun Wu, Daiji Okamura & Juan Carlos Izpisua Belmonte 

Abstract

This protocol describes a method to graft conventional primed pluripotent stem cells (PSCs) and region-selective PSCs to different locations of post-implantation mouse epiblast followed by in vitro embryo culture. Compared to conventionally used teratoma formation assay, epiblast grafting offers a more relevant in vivo test for evaluating the primed PSCs’ pluripotency and uniquely serves to distinguish different flavors of primed PSCs that harbor distinct spatial properties. Moreover, the observation that grafted human rsESCs could enter into early mouse embryogenesis provides a novel way of studying human development.

Introduction

Two temporally distinct pluripotent stem cells (PSCs) have been derived in mouse: “naïve” embryonic stem cells (ESCs) (1,2) and “primed” epiblast stem cells (EpiSCs) (3,4). They differ from each other in molecular signatures and phenotypic properties (5). As embryo development is a dynamic p

scientificprotocols

Authors: Kyung Duk Koh, Jay Hesselberth & Francesca Storici 

Abstract

Ribonucleotides are now widely considered as the most frequently incorporated non-canonical nucleotides in genomic DNA. Here, we describe an approach, ribose-seq, to prepare DNA libraries for next-generation sequencing to map ribonucleotide incorporation into DNA. By specifically targeting and capturing the unique ends of alkali-derived 2′,3′-cyclic monophosphate or 2′-monophosphate from ribonucleotides embedded in DNA, ribose-seq libraries are constructed. Upon high-throughput sequencing and analysis, the distribution and identity of ribonucleotides in the genome could be determined as recently reported using yeast as a model organism1. Ribose-seq could potentially be applied to any cell type of any organism to allow profiling of ribonucleotide incorporation into genomic DNA.

Introduction

Ribonucleotides, also known as ribonucleoside 5′-monophosphates (rNMPs), which are normally monomers of RNA, have been found to be the m

scientificprotocols

Authors: Chilamakuri C Sekhar Reddy, Khader Shameer, Offmann Bernard & Ramanathan Sowdhamini 

Abstract

Protein domains are the structural and functional units of proteins. The ability to parse proteins into different domains is important for effective classification, understanding of protein structure, function and evolution and is hence biologically relevant. Several computational methods are available to identify domains in the sequence. Domain finding algorithms often employ stringent thresholds to recognize sequence domains. Identification of additional domains can be tedious involving intense computation and manual intervention but can lead to better understanding of overall biological function. In this context, the problem of identifying new domains in the unassigned regions of a protein sequence assumes a crucial importance. We report the availability of a convenient server for the domain prediction in unassigned regions in proteins (PURE) which can be accessed at http://caps.ncbs.res.in/PURE

scientificprotocols

Authors: Yan Zhang 

Introduction

This protocol describes a detailed single cell microinjection technique to both attached and suspended cells, including cultured primary cells, cell lines and protozoan. The procedure optimizes injection parameters and performs serial preliminary control experiments to ensure least cellular stress and highest delivery efficiency. This procedure has been successfully applied to deliver exogenous proteins/cDNA constructs/drugs into transfection-challenged cells. With the precisely controlled delivery dosage and subcellular location, microinjection has been used in the studies of primary cultured cells, transgenic animal production, in vitro fertilization and RNA inference. A single injection to one cell should be finished within 5 seconds.

Reagents

1. Phosphate-buffered saline (PBS) ▲ CRITICAL Use sterile PBS for all injection experiments.

• Paraformaldehyde 4% (w/v) in PBS ! CAUTION Stored at 4 ºC. Paraformaldehyde is toxic to human. Avoid contact and inhalat