Antibodies


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.

As part of our mission to publish as many “gold standard” laboratory techniques as possible, I’m happy this month to include set of protocols covering the use of bromodeoxyuridine (BrdU) incorporation as a means of measuring DNA replication. The number of cells going through the cell cycle and their rate of progression are important indicators of cell growth. BrdU is a thymidine analog, which gets incorporated into new strands of DNA in a replicating cell in place of thymidine. It can then be detected with anti-BrdU antibodies. August’s issue of CSH Protocols provides three protocols for this method, from Dean Jackson and Peter R. Cook:
Analyzing DNA Replication I: Labeling Animals, Tissues, and Cells with Bromodeoxyuridine (BrdU)
Analyzing DNA Replication II: Fixation and Processing of Tissues and Cells Labeled with Bromodeoxyuridine (BrdU)
Analyzing DNA Replication III: Antibody Labeling of Incorporated Bromodeoxyuridine (BrdU) in Tissues and Cells

Immunohistochemistry (the localization of proteins in a tissue by binding antibodies to specific antigens) is a technique where one protocol definitely does not fit all. Each model organism seems to have its own quirks, whether it be in the fixative used, the methods needed for antibody penetration, issues with autofluorescence or even just figuring out which cross-species antibodies work in a given system. To that end, we’ve been working on expanding our coverage of immunohistological protocols. The February issue of CSH Protocols brings methods for plant sections, using both avidin-biotin and alk-phos, as well as a method for whole-mount immunocytochemistry in Xenopus embryos from John Wallingford’s lab at the University of Texas (they provided the lovely cover image for this month).

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Our protocol on Preparation of GST Fusion Proteins has seen a great deal of activity on the site, so this month we’ve supplemented it with several other protocols and background articles. This should give a better set of tools to do GST pull-downs and Far Westerns. (more…)

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