Antibodies


Immunoimaging is rapidly developing from a merely descriptive technique into a set of methods and analytical tools that can be used to quantify and characterize an immune response at the cellular level. In the February issue of Cold Spring Harbor Protocols, Ian Parker and colleagues from the University of California, Irvine present Immunoimaging: Studying Immune System Dynamics Using Two-Photon Microscopy. The article outlines the hardware required for immunoimaging and discusses methods for quantitative analysis of multidimensional image stacks. As one of our featured articles for February, this overview is freely available to subscribers and nonsubscribers alike.

The issue also contains protocols from the same authors for a General Approach to Adoptive Transfer and Cell Labeling for Immunoimaging, Induction of an Immune Response for Imaging Antigen-Presenting Cell/T-Cell Interactions, In Situ Lymph Node Imaging and In Vivo Lymph Node Imaging.

New technologies and methods are spurring a renaissance in the study of organogenesis. Organogenesis, essentially the process through which a group of cells becomes a functioning organ, has important connections to biological processes at the cellular and developmental levels, and its study offers great potential for medical treatments through tissue engineering approaches. The January issue of Cold Spring Harbor Protocols features a method from Washington University’s Hila Barak and Scott Boyle for Organ Culture and Immunostaining of Mouse Embryonic Kidneys. The kidney is particularly interesting as it also serves as a model for branching morphogenesis. The protocol describes the isolation, culture and fluorescent immunostaining of mouse embryonic kidneys. As one of January’s featured articles, the protocol is freely available to subscribers and nonsubscribers alike.

Blood feeding mosquitoes transmit many of the world’s deadliest diseases, which are resurgent in developing countries and pose threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for disease control through the genetic manipulation of vector insects. To accomplish this, vector insects must be established as laboratory model organisms, allowing for a better understanding of their biology, and in particular, the genes that regulate their development. Aedes aegypti is a vector mosquito of great medical importance because it is responsible for the transmission of dengue fever and yellow fever. In the October issue of Cold Spring Harbor Protocols, Molly Duman-Scheel and colleagues present an overview of the background, husbandry, and potential uses of Ae. aegypti as a model species. Protocols are provided for culturing and egg collection, fixation and tissue preparation, whole mount in situ hybridization, immunohistochemical analysis and RNA interference in Ae. aegypti. This methodology, much of which is applicable to other mosquito species, is useful to both the comparative development and vector research communities.

This article series marks the latest entrant in Cold Spring Harbor Protocols’ long-running series on Emerging Model Organisms.

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.

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