Immunoprecipitation is a commonly used technique for isolating and purifying a protein of interest. An antibody for the protein is incubated with a cell extract, and the resulting antibody/antigen complex is pulled out of solution. The method used for preparation of the cell extract can be critical for the experiment’s success. The choice of lysis conditions must be tailored to the nature of the epitope recognized by the immunoprecipitating antibody. Lysis of Cultured Cells for Immunoprecipitation, featured in the August issue of Cold Spring Harbor Protocols provides instructions for the lysis of cells grown as monolayer cultures and cells grown in suspension. The protocol offers a detailed comparison between different commonly used lysis buffers and protease inhibitor cocktails, as well as a guide to preparing a general protease inhibitor cocktail. As one of our featured articles, the protocol is freely available to subscribers and non-subscribers alike.

Cold Spring Harbor Laboratory Press’ new Drosophila Neurobiology laboratory manual covers the three main approaches taught in the CSHL course: studying neural development, recording and imaging the nervous system, and studying behavior. The featured electrophysiology paper is part of the recording/imaging section, while the second featured article in the July issue of Cold Spring Harbor Protocols comes from a neural development chapter.

The larval Drosophila brain has been a valuable model for investigating the role of stem cells in development. These neural stem cells, called “neuroblasts,” have provided insight into the role of cell polarity in influencing cell fate. Identifying neuroblasts and their progeny requires a method capable of recognizing cell polarity and cell fate markers. Immunofluorescent Staining of Drosophila Larval Brain Tissue, provided by Cheng-Yu Lee and colleagues, describes procedures for the collection and processing of Drosophila larval brains for analysis of these markers. Neuroblasts are identified via immunolocalization, the use of labeled antibodies that specifically bind the marker proteins of interest. As one of our featured articles, it is freely available to subscribers and non-subscribers alike.

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.

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 series on emerging model organisms continues this month, bringing you a set of articles on two systems that may be new to you, and one that’s a long-time classic.

Marianne Bronner-Fraser and colleagues have written up a guide to using the Sea Lamprey, Petromyzon marinus in the laboratory. The unique evolutionary position of the lamprey makes it a fascinating animal for comparative studies, and there are also lamprey-specific systems that are being investigated, like its variable lymphocyte receptor-mediated immune system. Protocols for culturing embryos, microinjection of RNA and morpholinos, DiI cell labeling, whole-mount in situ hybridization and immunohistochemistry are available.

Nipam Patel’s group at Berkeley brings us a look at the amphipod crustacean Parhyale hawaiensis. This crustacean is extremely amenable to laboratory studies, producing large amounts of embryos year round. The establishment of the segemented body plan is a particular area of interest for studies of P. hawaiensis. Protocols are provided for fixing and dissecting embryos, injection with fluorescent dyes, antibody staining and in situ hybridization.

Rusty Lansford and colleagues have written up their methods for using the classic developmental biology system, the Japanese Quail, Coturnix coturnix japonica. Their transgenic system is a big breakthrough, and deserves its own blog article, which I’ll post next week.

One of the great advantages of CSH Protocols over other online methods sources is that we have access to material from Cold Spring Harbor Laboratory’s cutting edge laboratory courses. November’s issue of CSH Protocols features material from the Molecular Embryology of the Mouse Course (as noted earlier), and the first set of many upcoming protocols from the Proteomics Course.

Course directors Andrew Link and Josh LaBaer have done a stellar job putting together a new laboratory manual based on the course. Proteomics will be out in December, but in the meantime, we’re publishing methods from the manual in advance in CSH Protocols. November’s issue brings a set of seven protocols covering Construction of Nucleic Acid Programmable Protein Arrays (NAPPA).

NAPPA differs from other protein array approaches in that proteins are translated in situ on the array surface, removing the need for individual protein purification. From the introduction:

This method uses cell-free extracts that transcribe and translate DNA into proteins which are then captured in situ, thus converting cDNA copies of genes into the desired target proteins. Instead of printing proteins at each feature of the array, the cDNA molecules for the corresponding genes that produce desired proteins are affixed to the array. Chemical treatment of glass slides and DNA isolation can be performed in advance and stored. The plasmid DNA can then be printed to make NAPPA slides, which can be stored dry for use. For experiments, NAPPA slides are expressed followed by detection of proteins and DNA using antibodies and stains.

Protocols are available for preparing slides and cultures, isolating DNA, labeling and arraying DNA, expressing proteins, detecting proteins and detecting DNA.