content Protocols

Category: Bacteria Genetics Protocols

Protocol for the identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection. Protocol describes ways in which difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances. - [Read Identification of Positive GATEWAY Expression Clones Protocol]

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Protocol for a rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. - [Read Identifying Transformed Arabidopsis thaliana Seedlings Following Floral Dip Transformation Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain 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: Microscopy Protocols: Electron Microscopy Protocols

In recent years, the increased sensitivity of electron detectors and the availability of low-vacuum or variable-pressure systems have allowed imaging of fresh tissue samples without the need for fixation, drying, and coating. This obviously saves a lot of time, although the image quality may not be as good as that obtained from fixed samples. However, for most applications that tend to be at a relatively low magnification, the quality can be as good as that obtained from fixed samples. - [Read Imaging of Fresh Arabidopsis Tissues in the Scanning Electron Microscope]

Category: Plant Biology Protocols

In recent years, the increased sensitivity of electron detectors and the availability of low-vacuum or variable-pressure systems have allowed imaging of fresh tissue samples without the need for fixation, drying, and coating. This obviously saves a lot of time, although the image quality may not be as good as that obtained from fixed samples. However, for most applications that tend to be at a relatively low magnification, the quality can be as good as that obtained from fixed samples. - [Read Imaging of Fresh Arabidopsis Tissues in the Scanning Electron Microscope Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Using confocal laser-scanning microscope & GFP fusion proteins in time-lapse imaging to visualize the behavior of organelles and to track membrane-bound transport intermediates that bud off from organelles. Practical issues related to construction & expression of GFP fusion proteins are discussed. Essential for optimizing the brightness and expression levels of GFP fusion proteins so that intracellular membrane-bound structures containing these fusion proteins can be readily visualized. - [Read Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells]

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Protocol describes how antibodies are bound to protein A or protein G beads and are then crosslinked to the matrix via a bifunctional coupling reagent called dimethyl pimelimidate (DMP). - [Read Immunoaffinity Purification: Coupling Antibodies to Protein A or G Bead Columns Protocol]

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: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents.  Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags.  Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. - [Read Immunoblotting: Antigen Detection Using Chemiluminescence Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents.  Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. - [Read Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

For immunoblotting experiments, it is often important to compare the total amount of an antigen from many different sources or to learn if a particular source has the antigen under study.  In the approach described here, tissue cultures, bacteria, yeast cells, tissues, and other sources of antigens are disrupted directly in an electrophoresis sample . - [Read Immunoblotting: Preparing Cell Lysates Protocol]

Category: Western Blot Protocols

Protein immunoprecipitation can be a useful preparative step for immunoblotting.  For very rare proteins, the protein of interest can be purified and concentrated by standard immunoprecipitation techniques before immunoblotting.  In addition, protein-protein interactions can be tested with an immunoprecipitating antibody that is specific for one protein of a complex and an immunoblotting antibody that is specific for a second member of the complex. - [Read Immunoblotting: Preparing Immunoprecipitated Proteins Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

Protocol describes how proteins are mixed with a sample buffer and prepared for electrophoresis on standard Tris/glycine SDS-polyacrylamide gels. - [Read Immunoblotting: Preparing Protein Solutions Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

The transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using a semi-dry method is achieved by placing the gel next to a piece of nitrocellulose filter.  This sandwich is placed directly between two plate electrodes, and the proteins are then transferred from the gel onto the filter. - [Read Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]

Category: Western Blot Protocols: General Western Blot Methods Protocols

Transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using the submerged method is achieved by placing the gel next to a piece of nitrocellulose filter, submerging this sandwich in a large volume of transfer buffer in a transfer tank, and running current from one side of the transfer tank to another.  The proteins are then eluted by transferring them from the gel onto the filter. - [Read Immunoblotting: Submerged Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]

Category: Immunology: Immunofluorescence Protocols

This protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells. Western blot technology can be used as an alternative (see Detection of RNAi-Induced Protein Knockdown in Mammalian Cells by Western Blotting). - [Read Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol]

Category: RNAi Technology Protocols: RNAi Mammalian Cells Protocols

Protocol describes the use of a specific antibody that recognizes the targeted gene product to detect RNAi-induced gene knockdown in mammalian cells.  Western blot technology can be used as an alternative. - [Read Immunofluorescence Detection of RNAi-Induced Protein Knockdown in Mammalian Cells Protocol]

Category: Immunohistochemistry Protocols: Cryosectioning Protocols

This protocol describes methods for the fixation, cryosectioning, and immunohistochemical detection of proteins in embryonic and adult zebrafish eyes. The methods described can easily be adapted for use on other zebrafish tissues. - [Read Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol]

Category: Laboratory Model Organisms Protocols

Protocol describes methods for the fixation, cryosectioning, and immunohistochemical detection of proteins in embryonic and adult zebrafish eyes. The methods described can easily be adapted for use on other zebrafish tissues. - [Read Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol]

Category: Immunology: Immunoprecipitation Protocols

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. - [Read Immunoprecipitation: Denaturing Lysis Protocol]

Category: Bacterial Cell Culture Protocols

In this protocol, bacterial cells are lysed by being subjected to short, intense treatments with ultrasound, which breaks the cell walls and shears the DNA into sizes that will not affect the viscosity of the samples. Note that this method causes some denaturation of the samples. The resulting lysate is ready for preclearing. - [Read Immunoprecipitation: Lysing Bacteria by Sonication Protocol]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]