Hier finden Sie ausgesuchte Protokolle zu unterschiedlichen Methoden. Diese Liste wird regelmäßig ergänzt und ausgebaut.
1. Immunoblotting and Immunodetection
Immunoblotting (western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. This unit provides protocols for all steps, starting with solubilization of the protein samples, usually by means of SDS and reducing agents. Following solubilization, the material is separated by SDS-PAGE and the antigens are electrophoretically transferred to a membrane, a process that can be monitored by reversible staining with Ponceau S. The transferred proteins are bound to the surface of the membrane, providing access to immunodetection reagents. After nonspecific binding sites are blocked, the membrane is probed with the primary antibody and washed. The antibody-antigen complexes are tagged with horseradish peroxidase or alkaline phosphatase coupled to a secondary anti-IgG antibody, and detected using appropriate chromogenic or luminescent substrates. Finally, membranes may be stripped and reprobed. Curr. Protoc. Mol. Biol. 83:10.8.1-10.8.28. © 2008 by John Wiley & Sons, Inc.
Immunohistochemistry is a vastly diverse and essential method for localization of proteins in cells and tissues. This unit presents methods for labeling proteins in suspension and adherent cultures and in tissue sections, using detection methods for both fluorescence and bright-field microscopy. Choices of antibodies and detection methods are discussed, and detailed troubleshooting guidelines are provided. Curr. Protoc. Mol. Biol. 81:14.6.1-14.6.23. © 2008 by John Wiley & Sons, Inc.
3. Growth and Manipulation of Yeast
Yeast cultures can be grown, maintained, and stored in liquid media or on agar plates using techniques similar to those for bacterial cultures. This unit describes culture conditions for these basic techniques. Additional methods describe determination of yeast mating type, diploid construction, sporulation, tetrad dissection, and random spore analysis. Curr. Protoc. Mol. Biol. 82:13.2.1-13.2.12. © 2008 by John Wiley & Sons, Inc.
4. Analysis of Protein Tyrosine Phosphatases and Substrates
Protein tyrosine phosphorylation is a reversible post-translational modification that is essential for life in eukaryotic cells. The combinatorial action of both protein tyrosine kinases and protein tyrosine phosphatases (PTPs) determines the net level of cellular tyrosine phosphorylation. This unit discusses methods to determine the level of protein tyrosine phosphatase activity and methods for discovering novel substrates for protein tyrosine phosphatases. Curr. Protoc. Mol. Biol. 91:18.16.1-18.16.17. © 2010 by John Wiley & Sons, Inc.
5. ChIP-Seq: A Method for Global Identification of Regulatory Elements in the Genome
This unit describes ChIP-Seq methodology, which involves chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (Seq), and enables the genome-wide identification of binding sites of transcription factors (TFs) and other DNA-binding proteins. The process is initiated by cross-linking DNA and DNA-bound proteins. Subsequently, chromatin is isolated from nuclei and subjected to sonication. An antibody against a specific TF or DNA-binding protein is then used to immunoprecipitate specific DNA-TF complexes. ChIP DNA is purified, sequencing adapters are ligated, and 30- to 35-nucleotide (nt) sequence reads are generated. The sequence of the DNA fragments is mapped back to the reference genome for determination of the binding sites. Curr. Protoc. Mol. Biol. 91:21.19.1-21.19.14. © 2010 by John Wiley & Sons, Inc.
6. Targeted Metabolomics
The metabolome is the terminal downstream product of the genome and consists of the total complement of all the low-molecular-weight molecules (metabolites) in a cell, tissue, or organism. Metabolomics aims to measure a wide breadth of small molecules in the context of physiological stimuli or disease states. Metabolomics methodologies fall into two distinct groups: untargeted metabolomics, an intended comprehensive analysis of all the measurable analytes in a sample including chemical unknowns, and targeted metabolomics, the measurement of defined groups of chemically characterized and biochemically annotated metabolites. The methodologies considered in this unit focus on the processes of conducting targeted metabolomics experiments, and the advantages of this general approach are highlighted herein. This unit outlines procedures for extracting nitrogenous metabolites (including amino acids), lipids, and intermediary metabolites (including TCA cycle oxoacids) from blood plasma. Specifically, protocols are described for analyzing these metabolites using targeted metabolomics experiments based on liquid chromatography−mass spectrometry. Curr. Protoc. Mol. Biol. 98:30.2.1-30.2.24. © 2012 by John Wiley & Sons, Inc.