fusion Protocols

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Category: Immunology: Immunoprecipitation Protocols

Protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG. - [Read Epitope Tagging of Recombinant Proteins Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocols for gene expression and protein localization in Arabidopsis. Includes: Detection of the native protein; Detection of a recombinant version; Immunofluorescence detection in Arabidopsis protoplasts; Isolation of Arabidopsis seedling protoplasts; Subcellular localization of GUS-fusion proteins in Arabidopsis seedlings; Localization of Arabidopsis proteins with GUS in situ enzyme assay. - [Read Gene Expression and Protein Localization in Arabidopsis Protocols]

Category: Molecular Model Organisms: Yeast Two-Hybrid Screen Protocol

Detailed protocols and methods for GST protein purification. Vesicle Trafficking, Stanford. - [Read GST Fusion Protein Prep]

Category: Molecular Model Organisms: Yeast Two-Hybrid Screen Protocol

GST Fusion Protein Purification from Yeast. Lysis Buffer recipe for yeast, glutathione bead method for GST purification. Dohlman Lab - [Read GST Fusion Protein Purification from Yeast]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Protein Purification and Isolation Protocols

Protocol for GST fusion protein purification from Yeast. - [Read GST Fusion Protein Purification from Yeast Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Developed PCR-based single molecule haplotyping methods that enable both surveys for novel inversion variants, and population-scale genotyping of known inversions - [Read Haplotype-Fusion PCR 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: 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: Microscopy Protocols: Live-Cell Imaging Protocols

GFP serves as a molecular marker that can be imaged dynamically in living cells, both in its native form & as a fusion to other proteins. For GFP imaging, plants present the challenge of autofluorescence from chlorophyll, lignified cell walls, vacuolar contents, and other cell materials, all of which can obscure the GFP signal. Maximizing the signal-to-noise ratio is a major concern, and careful consideration should be given to the choice of tissue imaged, GFP expression level, etc. - [Read Live-Cell Imaging of GFP in Plants]

Category: Antibody Protocols: Antibody Production Protocols

Monoclonal Antibody Production. Includes: Mouse Immunization; Bleeding Mice; Hybridoma Fusion; Assaying for Positive Clones; Expanding Positive Clones. - [Read Monoclonal Antibody Production]

Category: Antibody Protocols: Antibody Production Protocols

Protocol for monoclonal antibody production fusion. Fusion is an important procedure to produce monoclonal antibodies (MoAb). - [Read Monoclonal Antibody Production Fusion Protocol]

Category: Cell Biology and Cell Culture: Cell Line Culture Conditions

PC12 Cell Culture and Fusion Methods. Yu-li Wang - [Read PC12 Cell Culture and Fusion]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage {lambda} encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

This rapid method is used to screen bacteriophage {lambda}gt11 clones for the production of immunodetectable fusion proteins. After optimizng the conditions of infection and induction, the method can be used to produce preparative amounts of a fusion protein. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda}: Lytic Infection]

Category: Immunology: T Cell Protocols

T cell hybridomas can be obtained by fusing activated T cells with tumor cells. A heterogeneous population of hybridomas can be cloned by limiting dilution to obtain hybridomas that express specificity to one T cell receptor (TCR). Protocol describes cell fusion and selection of T cell hybridomas. A protocol is provided for screening of T cell hybridomas for expression of the CD3-TCR complex by flow cytometry analysis. - [Read Production of Mouse T Cell Hybridomas Protocol]

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the purification of GST-fusion proteins E. coli. (pGEX vector). - [Read Purification of GST-fusion proteins E. coli. (pGEX vector) Protocol]

Category: DNA Protocols: cDNA Library Protocols: cDNA Library Construction Protocols

An expression library constructed in a bacteriophage {lambda} vector is plated on an appropriate E. coli strain in the absence of isopropylthio-ß-D-galactoside (IPTG). After 2-4 hours, the plates are moved to 37°C (to stabilize any fusion proteins that are temperature sensitive), and filters impregnated with IPTG are laid on top of the developing plaques. - [Read Screening Expression Libraries Constructed in Bacteriophage Lambda Vectors Protocol]

Category: Antibody Protocols: Antibody Production Protocols

Protocol for steps in hybridoma production. Includes: SIMPLE METHOD: BY FROM GIBCO the HAT and HT concentrate; INSTRUMENTS FOR SPLENECTOMY OF ONE MICE; PREPARATION FOR CELL FUSION; CELL FUSION; SCREENING THE CLONES; RECLONING; ASCITIC FLUID PRODUCTION; PREPARATION OF PERITONEAL MACROPHAGES. - [Read Steps in Hybridoma Production Protocol]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

GUS is used as a tag to address nuclear localization whereas GFP is more versatile. GFP is detectable directly in living cells, GUS is only detected indirectly by staining of fixed tissue which may lead to artifacts or may obscure problems with protein solubility. In this protocol, protein localization is routinely assayed after particle-mediated transient transformation of onion epidermal cells. With this method it can be determined rapidly whether a given fusion protein is active and.... - [Read Subcellular Localization of GUS- and GFP-Tagged Proteins in Onion Epidermal Cells]

Category: Yeast Protocols: Yeast Protein Protocols: Yeast Immunoprecipitation Protocols

Protocol for TAP fusion protein purification from Yeast. - [Read TAP Fusion Protein Purification from Yeast Protocol]

Category: Neuroscience Protocols: Patch Clamping Protocols

The cell-attached capacitance recording technique is a powerful technique that has been successfully used to resolve single vesicle fusion and fusion pore conductance.  This technique, however, has not been applied to synapses because of the difficulty in accessing release sites.  Here, we developed a technique to expose release sites in a large nerve terminal, the calyx of Held, which contains clear-core glutamatergic vesicles. - [Read The Cell Attached Capacitance Recording Technique]

Category: Yeast Protocols: Yeast Two-Hybrid Systems Protocols

Protocol describes how to generate a plasmid construct (pBAIT) that expresses a target protein fused to the bacterial LexA protein. PBAIT is cotransformed into yeast with a lexAop-lacZ reporter plasmid carrying the bacterial lacZ gene under the control of the lexA operator. The recipient yeast strain contains a chromosomally integrated leu2 reporter gene, also under the control of the lexA operator. - [Read Two-hybrid Systems Stage 1: Characterization of a Bait-LexA Fusion Protein Protocol]