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Studying Early Hematopoiesis Using Avian Blastoderm Cultures

The process of hematopoiesis occurs very early during embryogenesis, with the appearance of red blood cells within the yolk sac (1,2). It is at the gastrula stage when hematopoietic stem cells arise from newly formed mesoderm (3,4). Only mesoderm cells found within the extraembryonic area opa ...

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Isolation and Culture of Mouse Germ Cells

The isolation and culture of germ cells has allowed both the analysis of gene expression in these cells as well as studies of their behavior and growth requirements. Some of the factors found to be important for the in vitro culture of germ cells have subsequently been found to be physiologically rele ...

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Analysis of Hyaluronan Using Biotinylated Hyaluronan-Binding Proteins

In this chapter, we describe the preparation and use of b-PG, a biotinylated complex that specifically binds hyaluronan (1,2). The b-PG is derived from cartilage and consists of a trypsin fragment of the proteoglycan core protein and one of the link proteins. Because of its ability to bind to hyaluron ...

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Cadherin-Mediated Cell-Cell Interactions

Cadherins constitute a family of transmembrane glycoproteins (Table 1) that mediate cell-cell adhesion by their ability to self-associate (1–4). For example, E-cadherin binds only to E-cadherin and not to N-cadherin. The homotypic interaction of cadherins is important for the sorting ...

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Microinjection of Fluorescently Labeled -Actinin into Living Cells

The introduction of microinjection of trace amounts of a fluorescently labeled protein (1,2) into cultured cells leads to its incorporation into the cell’s pool of endogenous protein. Provided the microinjected protein has retained the properties of the native protein, it will become i ...

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Genetic-Engineered Models of Skeletal Diseases II: Targeting Mutations into Transgenic Mice Chondrocytes

The manipulation of mice genome is a powerful technique to analyze biological processes that cannot be studied in tissue culture (1). The understanding of many developmental processes is today improving because of such techniques. This is also changing the way human genetic diseases are s ...

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Genetic-Engineered Models of Skeletal Diseases I: Collagen Type X

Formation of the vertebrate skeleton is an intricately orchestrated, multistep process. Mechanisms underlying skeletogenesis need to account for the global specification of skeletal pattern, commitment of cells to skeletal and hematopoietic lineages, local control of skel ...

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Pax3 and Vertebrate Development

Pax3, a transcription factor expressed in the developing embryo, is a critical factor for the proper formation of the mammalian nervous, cardiovascular, and muscular systems. In the mouse, spontaneous mutations in Pax3 resulting in complete loss of function have provided important mod ...

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Transgenic Mouse Models of Craniofacial Disorders

Disorders affecting craniofacial development represent a large fraction of birth defects (1). The advent of transgenic and gene knockout technology has led to the identification of a variety of genes that have roles in craniofacial development. These technologies, together with the t ...

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Selection for Soluble Proteins via Fusion with Chloramphenicol Acetyltransferase

The low solubility of a protein is one of the most frequent impediments for its structural and functional analysis and, on a more practical aspect, for its application as an industrial enzyme. The reason for low solubility can lie in low conformational stability (1), in a high number of surface-expos ...

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Proside: A Phage-Based Method for Selecting Thermostable Proteins

For many applications proteins must retain their function for a long time and under a wide range of conditions, sometimes at elevated temperatures. However, the stability of natural proteins is often very low, just sufficient to ensure proper functioning under cellular conditions (1). It is s ...

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Minimization of Proteins by Random Fragmentation and Selection

Protein-protein interactions are involved in most biological processes and are an important target for drug design. Over the past decade, there has been an increased interest in the design of small molecules that mimic functional epitopes in protein-protein interactions. However, the ...

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Solid-Phase Screening Using Digital Image Analysis

It is often possible to screen libraries of variant enzymes directly on agar plates. Colony-based solid-phase screening is an attractive option because of its relative ease and high throughput compared to liquid-phase screening in multi-well plates. As with any high throughput screeni ...

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Screening for Thermostability

Enzyme thermostability is a property of great importance in the era of designed biocatalysts. While enzymes are capable of catalyzing reactions with exquisite specificity and selectivity, they are often limited by insufficient stability. Improvements in enzyme activity throu ...

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High-Throughput Screening of Mutant -Amylase Libraries for Increased Activity at 129C

We describe a high throughput screening setup for measuring α-amylase activity at temperatures far above the boiling point of water. The system consists of a sealed aluminum 384-well assay plate incubated between two preheated aluminum blocks. Samples consisting of starch solution and ...

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High-Throughput Carbon Monoxide Binding Assay for Cytochromes P450

Cytochromes P450 are a superfamily of enzymes that catalyze the monoxygenation of a variety of substrates, including aliphatic and aromatic compounds (1). They contain a noncovalently bound protoporphyrin IX, giving these enzymes characteristic spectral properties. This heme ...

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High-Throughput Screen for Aromatic Hydroxylation

The oxidation of aromatic compounds is important in producing chemical intermediates for the chemical and pharmaceutical industries (1,2). Conventional aromatic oxidation reactions are prone to byproduct formation and often require heavy-metal catalysts, extremes of tem ...

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Colorimetric Screen for Aliphatic Hydroxylation by Cytochrome P450 Using p-Nitrophenyl-Substituted Alkanes

The United States consumes approximately 16–17 million barrels of crude oil per day, and the majority is used for electricity, heating, and transportation fuel (1). The main constituents of crude oil are linear aliphatics. Clearly, the selective hydroxylation of alkanes to more valuable pr ...

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High-Throughput Tetramethylbenzidine (TMB) Screen for Peroxidases

Peroxidases catalyze the decomposition of hydrogen peroxide coupled to the oxidation of a variety of organic and inorganic substrates. Produced by a broad variety of natural sources, most peroxidases use heme or vanadium as a cofactor at the redox active site, while some bacterial peroxid ...

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High-Throughput Screens Based on NAD(P)H Depletion

Screening conditions should simulate the final application as closely as possible. This is especially a challenge when chromophore-free (e.g., aliphatic) substrates are used and no simple and reliable high-throughput method for quantitative analysis of the respective reaction ...

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