Proteins and Proteomics

The use of recombinant proteins, antibodies, small molecules, or nucleic acids as affinity reagents is a simple yet powerful strategy to study the protein/bait interactions that drive biological processes. Analysis via mass spectrometry rather than western blotting extends the identification of interactors, often allowing detection of thousands of proteins from complex mixtures. But this increased sensitivity can lead to problems distinguishing specific interactions from background noise. In the March issue of Cold Spring Harbor Protocols, Shao-En Ong from the Broad Institute of MIT and Harvard presents Unbiased Identification of Protein/Bait Interactions Using Biochemical Enrichment and Quantitative Proteomics. This method uses quantitative proteomics approaches to compare enrichment with the bait of interest against samples using control baits to allow sensitive detection and discrimination of specific protein/bait interactions. As one of March’s featured articles, it is freely available to subscribers and non-subscribers alike.

The enzyme-linked immunospot (ELISPOT) assay is considered by many to be the gold standard for monitoring cellular immune responses. The method is highly sensitive, quantitative, easy to use and amenable to high throughput screening. Until recently, the ELISPOT assay has been limited to the characterization of only one single effector molecule. Since the maintenance of both IFN-gamma and IL-2 by pathogen-specific T cells has been linked to a more favorable clinical outcome in human immunodeficiency virus (HIV) and Leishmania infections, an ELISPOT assay able to characterize both these effector molecules would be helpful for monitoring immune responses to certain infectious agents. Nicole Bernard and colleagues from the McGill University Health Centre present a protocol for Dual-Color ELISPOT Assay for the Simultaneous Detection of IL-2 and/or IFN-gamma Secreting T Cells in the January issue of Cold Spring Harbor Protocols. As interest in multifunctional T-cell monitoring in human diseases grows, this method is likely to be extensively used. The protocol is one of January’s featured articles, and is freely available to subscribers and non-subscribers alike.

Live cell imaging techniques are driving a revolution in biological research. Instead of viewing dead tissues and cells fixed at a particular stage of activity, scientists can now visualize dynamic changes as they happen, permitting a better understanding of complete processes. The revolution has been fueled by the implementation of genetically encoded fluorescent proteins, the subject of the 2008 Nobel Prize in Chemistry.

The diverse array of applications benefiting from fluorescent proteins ranges from markers targeted at organelles and protein fusions designed to monitor intracellular dynamics to reporters of transcriptional regulation and in vivo probes for whole-body imaging and detection of cancer. Fluorescent proteins have enabled the creation of highly specific biosensors to monitor a wide range of intracellular phenomena, including pH and metal-ion concentration, protein kinase activity, apoptosis, membrane voltage, cyclic nucleotide signaling, and tracing neuronal pathways. In the December issue of Cold Spring Harbor Protocols, David Piston and colleagues present Fluorescent Protein Tracking and Detection: Fluorescent Protein Structure and Color Variants, a comprehensive overview of the wide variety of fluorescent proteins that are currently available. The article features more than twenty movies of different fluorescent proteins in action and is a great primer for planning imaging experiments. As one of December’s featured articles, it is freely available to subscribers and non-subscribers alike.

In addition, the same authors have also contributed Fluorescent Protein Tracking and Detection: Applications Using Fluorescent Proteins in Living Cells. This article provides some general tips for the practical aspects of using and imaging enhanced green fluorescent protein (EGFP) and newer members of the color palette, as well as quantitative imaging of fluorescent proteins and imaging of several fluorescent proteins at the same time. Finally, an overview is provided for the different types of biosensors that have been derived from flourescent proteins.

Both articles are adapted from the spectacular new manual, Live Cell Imaging: A Laboratory Manual, Second Edition which is due out by month’s end.

CSH Protocols December Cover

CSH Protocols December Cover

The tandem affinity purification (TAP) procedure was pioneered in yeast for the purpose of purifying and characterizing protein complexes and has since been adapted for use in many organisms, including mammalian systems. The TAP procedure involves two sequential affinity purification steps to avoid non-specific protein interactions, a common problem in identifying proteins in complexes. Bimolecular Affinity Purification (BAP): Tandem Affinity Purification Using Two Protein Baits, from Ezra Burstein and colleagues, presents a variation on the TAP procedure in which the affinity moieties are placed on two different proteins of a molecular complex to isolate or detect components present in the complex. This variation, called bimolecular affinity purification (BAP), is suited for the identification of specific molecular complexes marked by the presence of two known components. The article is featured in the November issue of Cold Spring Harbor Protocols and like all our featured articles, is freely available to subscribers and non-subscribers alike.

Glutathione-S-transferase (GST) fusion proteins are used in a wide variety of applications in the lab. GST was originally selected as a fusion moiety because it’s not sequestered in inclusion bodies when expressed in bacteria and it can be affinity-purified without denaturation. Purification is fairly straightforward process, and GST fusion proteins are routinely used for antibody generation and purification, protein-protein interaction studies, and biochemical analysis.

Protocols describing the use of GST fusion proteins are among our most popular, and November’s issue of Cold Spring Harbor Protocols brings two new sets of instructions, covering Preparation of Soluble GST Fusion Proteins and Preparation of Insoluble GST Fusion Proteins. These articles complement our already extensive coverage, which includes the previously featured (and still freely available) article Preparation of GST Fusion Proteins. Our readers also frequently access Identification of Protein-Protein Interactions with Glutathione-S-Transferase (GST) Fusion Proteins, GST Pull-down, Detection of Protein-Protein Interactions Using the GST Fusion Protein Pulldown Technique, Detection of Protein-Protein Interactions Using Far Western with GST Fusion Proteins, Far Western: Labeling GST Fusion Proteins, and Far Western: Probing Membranes.

RNA molecules interact with proteins to drive many cellular activities, including post-transcriptional processing of RNA, regulation of translation, and transport of RNA to name but a few. These ribonucleoprotein complexes are isolated by coimmunoprecipitation (co-IP), where a protein-specific antibody is used to purify the protein of choice and its associated complex members. Analysis of RNA-Protein Complexes by RNA Coimmunoprecipitation and RT-PCR Analysis from Caenorhabditis elegans gives step-by-step instructions for RNA co-IP from C. elegans whole-worm extracts. The protocol, from Christian Eckmann and colleagues at the Max Planck Institute of Molecular Cell Biology and Genetics, starts with the large-scale growth of worms and describes the preparation of whole-worm extracts, RNA co-IP, isolation of the purified RNA, and identification of specific genes through RT-PCR. As one of our featured articles for October, the protocol is freely available to subscribers and non-subscribers alike. Eckmann and colleagues have also contributed an accompanying article describing Analysis of In Vivo Protein Complexes by Coimmunoprecipitation from Caenorhabditis elegans.

Chromatin Immunoprecipitation (ChIP) is an invaluable method for studying the interactions between proteins and DNA on a genome-wide scale. ChIP can be used to determine whether a transcription factor interacts with a candidate target gene, and is used to monitor the presence of histones with posttranslational modifications at specific genomic locations. The results are often extremely useful for investigating the functions of specific transcription factors or histone modifications. In the September issue of Cold Spring Harbor Protocols, Michael Carey, Craig Peterson and Stephen Smale present Chromatin Immunoprecipitation (ChIP), an optimized protocol for use in mammalian cells. This is one of September’s featured articles, and like all our featured articles, it is freely available to subscribers and non-subscribers alike.

RNA-binding proteins play important roles in all aspects of RNA metabolism, particularly in the regulation of mRNAs and subsequent control of gene expression. RNA Immunoprecipitation (RIP), much like Chromatin Immunoprecipitation (ChIP), is a method for analyzing the interactions between proteins and nucleic acids. In the June issue of Cold Spring Harbor Protocols, Jesper Svejstrup and colleagues from the London Research Institute provide RNA Immunoprecipitation to Determine RNA-Protein Associations In Vivo, a detailed set of instructions for RIP analysis. Proteins and RNAs are cross-linked by formaldehyde treatment and immunoprecipitated. RNAs are then recovered and characterized by RT-PCR. The method is particularly useful for kinetic analysis of interactions at different timepoints and under different environmental conditions.

High-throughput whole-genome analysis is becoming a standard laboratory approach for investigating cellular processes. Next-generation sequencing is replacing microarrays as the technique of choice for genome-scale analysis, because it offers advantages in both sensitivity and scale. The June issue of Cold Spring Harbor Protocols features Native Chromatin Preparation and Illumina/Solexa Library Construction from Keji Zhao and colleagues at the National Heart, Lung and Blood Institute. The article describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Solexa/Illumina Genome Analyzer. Step-by-step instructions are given for purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.

Our second featured article for the May issue of Cold Spring Harbor Protocols is Systematic Monitoring of Protein Complex Composition and Abundance by Blue-Native PAGE, written by Harvey Millar and colleagues from the University of Western Australia. The article describes multiple experimental approaches using polyacrylamide gel electrophoresis (PAGE). Blue-native PAGE (BN-PAGE) allows a range of protein complexes to be visualized. When combined with sodium dodecyl sulfate PAGE (SDS-PAGE), the procedure can resolve the complexes and their subunits by their molecular weight. In conjunction with differential in-gel electrophoresis (DIGE), BN-PAGE can be used to quantify changes in protein complex abundance or subunit composition between different samples. A detailed methodology is provided for BN-PAGE, SDS-PAGE, and DIGE, and like all of our featured articles, it is freely accessible to subscribers and non-subscribers alike.

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