tagged Protocols

Category: Cytoskeleton Protocols: Actin Myosin Protocols

Phalloidin binds specifically to F-actin, and fluorescent-tagged phalloidin stains the actin skeleton in cells in a manner that is very close to the staining pattern seen using anti-actin antibody. - [Read Actin Staining in Fixed Yeast Cells Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

Category: Cytogenetics Protocols: Comparative Genomic Hybridization CGH Protocols

Jackie Ryan, CGH Protocols. Univ Davidson. Excellent Detailed CGH procedure. Tagged DNA Hybridization, Post Hybridization Wash, Post 3DNA Hybridization Wash. Fragmentation. - [Read CGH Protocols PDF]

Category: Yeast Protocols: Yeast Protein Protocols

This assay is performed to detect ubiquitylated proteins in yeast. Yeast that have been transformed with a vector expressing polyhistidine-tagged ubiquitin (Ub) under the control of a copper-inducible promoter are grown, induced with copper, and harvested. Total ubiquitylated proteins are then recovered by nickel-affinity chromatography, and specific proteins are detected by Western blotting. - [Read Detection of Ubiquitylated Proteins in Yeast Protocol]

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: E Coli Protocols: E coli Protein Protocols

Protocol for the expression and purification of His tagged proteins in E. coli. - [Read Expression and Purification of His Tagged Proteins in E. coli Protocol]

Category: Chromatography Protocols

His Tag Nickel Affinity Chromatography Protocol PDF. The Wallert and Provost Lab. Theory and Introduction: Ni-Affinity Chromatography uses the ability of His to bind nickel. Six histadine amino acids at the end of a protein (either N or C terminus) is known as a 6X His tag. Nickel is bound to an agarose bead by chelation using nitroloacetic acid (NTA) beads. Several companies produce these beads as His Tagged proteins are some of the most used affinity tags in today’s market. - [Read His Tag Nickel Affinity Chromatography Protocol PDF]

Category: Reporter Gene Assays: GFP Assay Protocols

Information and tips on how to use GFP-tagged proteins in cell biology - [Read How to Use GFP-Tagged Proteins in Cell Biology]

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: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol for immunoprecipitation of epitope-tagged proteins from yeast. - [Read Immunoprecipitation of Epitope-Tagged Proteins from Yeast Protocol]

Category: Signalling Signal Transduction Protocols

Protocol describes how tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay. Protocol includes: Translation of Xenopus Mos Kinase; Antibody to Antigen Binding; Protein A Sepharose to Antibody Binding; Kinase Reactions on Immunoprecipitated Material; Polyacrylamide Gel Analysis of Immunoprecipitates. Includes protocol hints. - [Read Kinase Assay Using In Vitro Translated Xenopus Mos Kinase]

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: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols

Protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons.  The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. - [Read Transfecting Cultured Hippocampal Neurons with an Actin-GFP Plasmid Protocol]

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Troubleshooting the immunoprecipitation procedure Roche. Tagged proteins not visible or only weakly visible on Western blot. High background or many nonspecific bands on gel or blot membrane. - [Read Troubleshooting the immunoprecipitation procedure IP and IB - Roche]

Category: Protein Protocols: Protein Ubiquitination Protocols

Ultimate His-UB Assay for Mammalian Cells. Tansey Lab Protocols. William P. Tansey. PREPARATION OF NI-NTA-AGAROSE, HARVESTING THE TRANSFECTION., ELUTION OF HIS-TAGGED PROTEINS. - [Read Ultimate His-UB Assay for Mammalian Cells]

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes the use of FLAG-epitope-tagged proteins for both small-scale analysis and large-scale coimmunoprecipitation of interacting proteins. When examining protein interactions, it is sometimes possible to immunoprecipitate an endogenous protein X directly, without using an epitope tag, if antibodies are available. The advantage of examining interactions of endogenously expressed proteins is that these are likely to be physiological and less likely to be an artifact of overexpression. - [Read Using FLAG Epitope-Tagged Proteins for Coimmunoprecipitation of Interacting Proteins Protocol]