procedure Protocols

Category: HPLC Protocols

HPLC Protocol. A general HPLC procedure to get you started on High Performance Liquid Chromatography. - [Read HPLC Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes a method for establishing short-term explant cultures of oesophageal mucosa. Adverse effects produced by exposure to radiation or test compounds can be detected as an inhibition of cell outgrowth. - [Read Human Oesophageal Culture Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

This elution procedure relies on disrupting the bonds between the antibody and the antigen. - [Read Immunoaffinity Purification: Eluting Antigens from Immunoaffinity Columns Protocol]

Category: Molecular Imaging: Immunofluorescence Microscopy

Immunofluorescence staining. Includes Background, Procedure, Key points and materials / methods. Grainger Lab. - [Read Immunofluorescence staining]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Protocol for immunofluorescent staining of human peripheral blood (LWB Procedure). - [Read Immunofluorescent Staining of Human Peripheral Blood (LWB Procedure) Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for in vitro splicing reactions in Drosophila KC or HeLa nuclear extracts. Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read In Vitro Splicing Reactions in Drosophila KC or HeLa Nuclear Extracts]

Category: Fungi Protocols: Neurospora Protocols

Procedure for an in vitro system to analyze light signal transduction in Neurospora crassa - [Read In Vitro System to Analyze Light Signal Transduction in Neurospora crassa]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol for in vitro transcription and translation using the coupled reticulocyte lysate system. This protocol is designed to test random samples on a protein gel. Scale up the reactions accordingly. Protocol includes: Procedure, Solutions, BioReagents and Chemicals and protocol hints. - [Read In Vitro Transcription and Translation Using the Coupled Reticulocyte Lysate System]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

Protocol outlines the general procedure and requirements for in vitro translation of CFTR and outlines some assays using in vitro translated product. Core glycosylation of CFTR occurs in the ER. An assay for this processing step requires the addition of microsomal membranes to the basic in vitro translation mixture. This protocol takes this into account. - [Read In vitro Translation Assays for CFTR]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Protocol by using the technique of Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+. Protocol includes: Preparation of Indo-1 and preparation of Ionomycin. - [Read Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+]

Category: RNAi Technology Protocols: RNAi Avian Embryos Protocols

Protocol describes a method for in ovo transfection of avian embryos with double-stranded RNA (dsRNA).  The dsRNA is injected into the spinal cord of the embryo.  Subsequent electroporation facilitates the cellular uptake of the dsRNA molecules.  It may be necessary to optimize the stage of the embryo and the electroporation procedure to improve the effectiveness of in ovo RNAi—cell competence changes with differentiation. - [Read Injection of dsRNA and Electroporation in Avian Embryos Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

The basis of this procedure is that two specific cell type preparations may be isolated, exposed separately to various compounds over a range of concentrations, and the cytotoxicity of these determined.  Parameters deemed indicative of a cytotoxic effect include a reduction in de novo protein synthesis and decreased glucose and fatty acid metabolism.  A cytotoxic effect may indicate that a chemical is likely to be nephrotoxic in vivo. - [Read Isolated Rat Glomeruli and Proximal Tubules]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes the isolation and culture of adult mouse cardiac myocytes from one heart. The isolation routinely yields approximately 1 million rodshaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from One Heart Protocol]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This procedure describes the isolation and culture of adult mouse cardiac myocytes from two or more hearts. Includes modifications for the digestion of two or more hearts in the same procedure and subsequent pooling of myocytes derived from the multiple hearts. The isolation procedure is performed by one or more technicians and routinely yields approximately 1 million rod-shaped myocytes per heart. - [Read Isolation of Adult Mouse Cardiac Myocytes from Two or More Hearts Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

The procedure for isolation of BAC DNA is scaled-up to accommodate 500-ml cultures, which, on average, yield 20-25 µg of purified BAC DNA. - [Read Isolation of BAC DNA from Large-scale Cultures Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Procedure is used to prepare DNA simultaneously from many different types of samples or tissues.  Although the DNA is generally too small (approx.  80 kb) for efficient construction of genomic DNA libraries, it gives excellent results in Southern hybridizations and PCRs.  Cultured aneuploid mammalian cells (2 x 107, e.g., HeLa cells) yield 100 µg of DNA in a volume of 1 ml. - [Read Isolation of DNA from Mammalian Cells by Spooling Protocol]

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

This procedure describes the isolation of resting B lymphocytes (B cells) from mouse spleens by using negative selection with anti-CD43 and anti-Mac-1/CD11b monoclonal antibodies coupled to magnetic microbeads. This strategy depletes non-B cells from a mixed population of splenocytes and relies on the fact that most mature leukocytes, with the exception of resting splenic B cells, express CD43. - [Read Isolation of Resting B Lymphocytes Protocol]

Category: DNA Microarray Protocols

This protocol describes the hybridization of a Cy labeled cDNA probe (mix of Cy3 and Cy5) onto coated slide spotted with PCR amplified cDNA. TIGR Microarray Protocols. - [Read Labeled Probe Hybridization Procedure]

Category: Molecular Biology Protocols: DNA Ligation Protocols

10 × tailing buffer. Tdt procedure. Terminal transferase and cloning. Powell and Gannon. Oxford Practical Approach Series. - [Read Ligation of DNA fragments by homopolymer tailing]

Category: Molecular Biology Protocols: DNA Ligation Protocols

Ligation with GUS vector and Transformation. Chemical Transformation of E. coli. Ligation Procedure for LigaFastâ„¢ Rapid DNA Ligation System (Promega). B. Beason, Rice University. - [Read Ligation with GUS vector and Transformation]

Category: Molecular Imaging: Electron Microscopy Protocols

Protocol for sectioning is on a separate page. 2. Standard Procedure To Section for Transmission Electron Microscopy, EM Center, Indiana University School ... List of standard EM Center procedures/protocols - [Read List of standard EM Center procedures/protocols]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Protocol describes the acquisition and processing of confocal fluorescent and bright field images of live cells, expressing cyan fluorescent protein(CFP) and/or yellow fluorescent protein (YFP), with a spinning disk confocal head on a Zeiss Axiovert 200 M microscope. This procedure is used to help determine if N- or Cterminal tagging of signaling molecules alters the steady state localization pattern of the signaling protein in question. - [Read Live Cell Spinning Disk Confocal Fluore Imaging of Cells- Colocalization of Fluorescent Protein Tags]

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

No special treatment is required to prepare a lysate for the active collection. The following procedure should be used for long-term storage of lambda clones in the archival collections. The phage are diluted in media containing 7% DMSO and frozen at -80 degrees C. - [Read Long Term Lambda Phage Storage Protocol]