Cold Spring Harbor protocols最新文献

Antibodies consist of unique variable heavy (V_H) and variable light (V_L) chains, and both are required to fully characterize an antibody. Methods to detect paired heavy and light chain variable regions (V_H:V_L) using high-throughput sequencing (HTS) have recently enabled large-scale analysis of complete functional antibody responses. Here, we describe an HTS computational pipeline to analyze paired V_H:V_L antibody sequences and obtain a comprehensive profile of immune diversity landscapes, including gene usage, antibody isotypes, and clonal lineage analysis. This protocol uses Illumina MiSeq 2 × 300-bp sequencing data and integrates with several different computational tools for flexible analyses of paired V_H:V_L gene repertoire data to enable efficient antibody discovery.

Chromatin immunoprecipitation (ChIP) is a well-characterized procedure used to reveal specific patterns of protein-DNA interactions and identify the binding sites of proteins on DNA. ChIP has been used to study many aspects of Drosophila biology, including neurobiology. This protocol describes in detail how to prepare cross-linked chromatin from Drosophila antennae and brains followed by immunoprecipitation (X-ChIP). We first describe tissue dissection, chromatin cross-linking with formaldehyde, quenching of the cross-linking, homogenization of tissues, and sonication for shearing the chromatin. Additionally, we describe how to optimize the sonication efficiency and fixation time and concentration using Drosophila brain samples as an example. These parameters are crucial for successful ChIP.

Antibody repertoire sequencing and display library screening are powerful approaches for antibody discovery and engineering that can connect DNA sequence with antibody function. Antibody display and screening studies have made a tremendous impact on immunology and biotechnology over the last decade, accelerated by technological advances in high-throughput DNA sequencing techniques. Indeed, bioinformatic analysis of antibody DNA library data has now taken a central role in modern antibody drug discovery, and is also critical for many ongoing studies of human immune development. Here, we describe current trends in antibody DNA library screening and analysis, and introduce a selection of protocols describing fundamental bioinformatic techniques to enable scientists to efficiently study antibody DNA libraries.

Chromatin immunoprecipitation (ChIP) is a common approach for studying the binding pattern of proteins on DNA sequences or the landscape of histones with different marks throughout the genome. ChIP is used on various organisms, including Drosophila This protocol provides a detailed overview of the immunoprecipitation portion of a ChIP procedure from samples of Drosophila nervous systems, specifically antennae and brains, that have already been fixed and sheared. These methods can be applied to other tissues of interest after optimizing for sample size and other relevant parameters.

The chromatin state plays an important role in regulating gene expression, which affects organismal development and plasticity. Proteins, including transcription factors, chromatin modulatory proteins, and histone proteins, usually with modifications, interact with gene loci involved in cellular differentiation, function, and modulation. One molecular method used to characterize protein-DNA interactions is chromatin immunoprecipitation (ChIP). ChIP uses antibodies to immunoprecipitate specific proteins cross-linked to DNA fragments. This approach, in combination with quantitative PCR (qPCR) or high-throughput DNA sequencing, can determine the enrichment of a certain protein or histone modification around specific gene loci or across the whole genome. ChIP has been used in Drosophila to characterize the binding pattern of transcription factors and to elucidate the roles of regulatory proteins in gene expression during development and in response to environment stimuli. This review outlines ChIP procedures using tissues from the Drosophila nervous system as an example and discusses all steps and the necessary optimization.

Amino acids in maize can exist in both a free and protein-bound state. While most amino acids are part of a protein backbone, a small percentage of them remain free and play important biological roles, serving as signaling molecules, nitrogen transporters, osmolytes, and precursors for multiple primary and secondary metabolites. Their levels vary widely especially in maize leaves, depending on the developmental stage and in response to environmental conditions. Therefore, accurate and reliable quantification of free amino acids (FAAs) is vital in any effort aimed at studying their response to developmental and environmental cues. In this protocol, we describe a robust, high-throughput method that quantifies the 20 proteogenic amino acids (i.e., those that can be incorporated into proteins) that are found in the free form in maize tissue. This method consists of three major parts: first, aqueous extraction of FAAs from maize tissue; second, separation, detection, and quantification of all 20 proteogenic amino acids using ultraperformance liquid chromatography-tandem mass spectrometry; and third, data analysis and processing using the MassLynx data analysis software, TargetLynx.

In cereal crops, seed quality is determined by the composition and levels of protein-bound amino acids, which account for ∼90% of the seed total amino acid content. In maize particularly, seed quality is affected by the low levels of lysine and tryptophan, two amino acids that humans and animals cannot synthesize and must obtain from the diet. The low levels of these two amino acids in seeds is due to the dominance of seed storage proteins, namely zeins, which are deficient in these two amino acids. Many efforts have been deployed to improve the nutritional composition of maize kernels (i.e., seeds). Still, the lack of high-throughput and inexpensive methods for the quantification of amino acids that are found within proteins has limited those efforts, especially when large populations are targeted. In this protocol, we describe a robust, efficient, and high-throughput method for the quantification of all 20 proteogenic (protein-forming) amino acids from a crude protein extract. The method consists of four major parts: first, release of the 20 proteogenic amino acids from the protein backbone through hydrolysis; second, aqueous extraction of the released amino acids; third, separation, detection, and quantification of the released amino acids using a multiple reaction monitoring-based ultraperformance liquid chromatography-tandem mass spectrometry detection; and fourth, data analysis and processing using the MassLynx data analysis software, TargetLynx.

Amino acid analysis is a vital part of analytical biochemistry. The increasing demand for low nitrogen fertilization and for plant-based diets with balanced amino acid levels and composition have made it crucial to develop reliable, fast, and affordable methods for analyzing amino acids in plants. As maize accounts for 43% of global cereal production, improving the amino acid composition of its kernels (i.e., seeds) is critically important for meeting the dietary requirements of humans and livestock. Moreover, amino acid quantification in maize leaves is necessary for improving yield prediction, stress sensing, and nitrogen use efficiency. Many amino acid quantification methods use reverse-phase high-pressure liquid chromatography and gas chromatography approaches to assess the amino acid content of maize tissues. Historically, these techniques involved the use of chemical derivatization, a chemical reaction that alters the properties of a compound to make it detectable or more sensitive to detection. Although accurate, these methods are time-consuming, expensive, and unsuitable for large populations. Here, we introduce two high-throughput methods for quantifying amino acids from large maize populations, such as those used for quantitative trait locus mapping, genome-wide association studies, and large mutant populations. Both methods use an ultraperformance liquid chromatography-tandem mass spectrometry instrument to quantify all 20 proteogenic amino acids in a maize tissue in a short run time. A dependable, affordable, and high-throughput method for quantifying amino acids in maize has important implications for assessing kernel quality, yield, and management efficacy, such as fertilizer usage and watering.

Fluorescence in situ hybridization (FISH) is a macromolecular recognition tool that uses RNA or DNA fragments combined with fluorophore- or digoxigenin-coupled nucleotides as probes to examine transcript localization through the presence or absence of complementary sequences in fixed tissues or samples under a fluorescent microscope. FISH technology has been highly effective for mapping genes and constructing a visual map of animal genomes. Here, we describe the application of FISH technology in the Aedes aegypti mosquito, where it is specifically used to localize receptor transcripts in gut tissues/organs. The methods presented highlight the synthesis of RNA probes and describe the 2-d process of incubating the tissues/organs with the RNA probes. We also describe tyramide signal amplification for improved signal detection.

Nervous system formation involves the specification of neuron identity, followed by precise circuit construction; this includes controlling the pattern and connectivity of the dendrite arbor. Drosophila dendritic arborization (da) neurons are a powerful experimental model for studying dendrite arbor differentiation mechanisms. da neuron dendrite arbors elaborate in two dimensions in the body wall, making it easy to visualize them with high resolution. Immunostaining is a conventional method to examine arbor pattern and the subcellular distribution of proteins. In addition, images acquired from immunostaining protocols can amplify weaker signals from fluorescent transgenic proteins and be used to quantify protein expression levels. This protocol describes a broadly applicable dissection, fixation, and immunostaining approach in Drosophila larvae.