CSH protocols最新文献

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. This approach, termed "Nucleic Acid Protein Programmable Array" (NAPPA), enables the simultaneous expression of proteins in microarray format without the need for individual protein purification. 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. This protocol describes the initial preparation of slides to be used in the method.

INTRODUCTIONThe ability to prospectively identify and characterize neural progenitor cells in vivo has been difficult due to a lack of cell-surface markers specific for these cell types. A widely used in vitro culture method, known as the Neurosphere Assay (NSA), has provided a means to retrospectively identify neural progenitor cells as well as to determine both their self-renewal capacity and their ability to generate the three primary cell types of the nervous system: neurons, astrocytes, and oligodendrocytes. Today, combined with the establishment of multiple transgenic mouse strains expressing fluorescent markers and advances in cell isolation techniques such as fluorescence-activated cell sorting (FACS), the NSA provides a powerful system to prospectively elucidate neural progenitor characteristics and functions. Here we describe methods for the isolation, culture, and differentiation of neural progenitors from the developing mouse and adult cortex.

INTRODUCTIONTomato (Solanum lycopersicum) is one of the most important vegetable plants in the world. It originated in western South America, and domestication is thought to have occurred in Central America. Because of its importance as food, tomato has been bred to improve productivity, fruit quality, and resistance to biotic and abiotic stresses. Tomato has been widely used not only as food, but also as research material. The tomato plant has many interesting features such as fleshy fruit, a sympodial shoot, and compound leaves, which other model plants (e.g., rice and Arabidopsis) do not have. Most of these traits are agronomically important and cannot be studied using other model plant systems. There are 13 recognized wild tomato species that display a great variety of phenotypes and can be crossed with the cultivated tomato. These wild tomatoes are important for breeding, as sources of desirable traits, and for evolutionary studies. Current progress on the tomato genome sequencing project has generated useful information to help in the study of tomato. In addition, the tomato belongs to the extremely large family Solanaceae and is closely related to many commercially important plants such as potato, eggplant, peppers, tobacco, and petunias. Knowledge obtained from studies conducted on tomato can be easily applied to these plants, which makes tomato important research material. Because of these facts, tomato serves as a model organism for the family Solanaceae and, specifically, for fleshy-fruited plants.

INTRODUCTIONCtenophores, or comb jellies, are a group of marine animals whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. Some characteristics are present in nearly all ctenophores, including biradial symmetry, comb rows composed of linked cilia, an apical sensory organ, and two tentacles bearing specialized adhesive cells. All ctenophores studied thus far have the same stereotyped cleavage program and go through a specific stage of development known as the cydippid larva, after which adult structures develop and diverge greatly among species; this is particularly useful for comparative studies. In some cases, gene expression patterns appear to be conserved. Of particular interest is the finding that some genes are expressed in regions of the ctenophore body that are not morphologically distinct from the adjacent areas. However, it has proven difficult to determine the orthology of some genes, possibly because of the extreme divergence of ctenophore representatives. This protocol describes how to isolate genomic DNA from ctenophores. The procedure can be applied to adult tissues, but it is best to use embryos and larvae. After washing and concentrating the embryos or larvae, DNA is extracted using DNAzol reagent, a guanidine-detergent lysing solution. The resulting DNA can be used for polymerase chain reaction (PCR) or other applications.

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. 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. This protocol describes a method for expression of the desired proteins in situ on the printed slides.

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. 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. This protocol describes antibody detection of the arrayed proteins.

INTRODUCTIONFunctional proteomics enables protein activities to be studied in vitro using high-throughput (HT) methods. Protein microarrays are the method of choice because they display many proteins simultaneously and require only small reaction volumes to assess function. Protein microarrays are typically used to (1) measure the abundance of many different analytes in a sample or (2) study the functions or properties of many proteins spotted on the array. Target protein microarrays are usually generated by expressing, purifying, and spotting the proteins onto a solid surface at very close spatial density. An alternative approach is to translate the proteins in situ on the array surface. 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. This protocol describes detection of DNA on arrayed slides to assess the amount of DNA captured.

INTRODUCTIONGrafting is agronomically important because one can combine desirable aboveground characteristics (such as fruit size) and underground characteristics (such as resistance to soil-borne diseases). This protocol describes the simplest way of grafting tomato plants using "top wedge grafting" or "cleft grafting." Potatoes, eggplants, and tobacco plants are closely related to tomatoes, and they can be grafted onto each other as well. Although the grafting of vegetable crops is still rare, this technique has been useful in reducing infections caused by pathogens, increasing resistance to drought, and enhancing nutrient uptake.

INTRODUCTIONThis protocol describes methods for determining the sex of an individual Astyanax mexicanus. Adult males and females differ most obviously in body shape and in form of the anal fin. However, if the sex of an adult cannot be determined by these differences, it can be assessed by testing the anal fin for the presence (or absence) of denticle or hook-like bony elements on the anterior fin rays of the anal fin. These features are observed as an opacity toward the anterior half of the male's anal fin, detected as described in the method presented here.

INTRODUCTIONThis protocol describes an alternative method for breeding Astyanax mexicanus, using in vitro fertilization. Sperm collected from the male and eggs collected from the female are placed in a Petri dish, and sperm are activated by the addition of fresh system water. The eggs are observed under low magnification for signs of fertilization, usually marked by the onset of cleavage.