ability Protocols

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Traditional animal models to quantify the degree of blood vessel formation are being replaced by cell culture assays that are easier to set up, statistically reliable and can be automated in a drug screening laboratory. These assays rely on the endothelial cells’ ability to form distinct blood-vessel-like tubules in an extracellular matrix where they can subsequently be visualized by fluorescence microscopy. - [Read An Image-Based Assay of Endothelial Cell Tube Formation as a Model of Angiogenesis]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

The assay for ß-galactosidase relies on the ability of the enzyme to catalyze the hydrolysis of ONPG (o-nitrophenyl-ß-D- galactopyranoside) to free o-nitrophenol, which absorbs light at 420 nm. In this protocol, extracts of cells transfected with a ß-galactosidase reporter plasmid are incubated with ONPG. - [Read Assay for ß-galactosidase in Extracts of Mammalian Cells]

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

The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription. - [Read Basic Information on In Vitro Transcription]

Category: Immunology: Chemotaxis

Chemotaxis Assay, Springer Lab. A chemotaxis assay's function is to assess whether a factor or molecule of interest has chemotactic activity on a motile cell type. Chemotaxis is the ability of a factor to cause the migration of a cell. The chemotactic assay is based on the creation of a chemical gradient of the chemotactic agent which will cause cells to migrate through the gradient towards the chemotactic agent. - [Read Chemotaxis Assay]

Category: Cell Biology and Cell Culture

This chemotaxis assay protocol is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. A chemotaxis assay can determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is then the ability of a protein to direct the migration of a specific cell. - [Read Chemotaxis Assay Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

It is possible that some cell lines have lost the ability to perform RNAi or that cells derived from certain tissues do not support RNAi. This reporter assay, for RNAi in mammalian cells, can be used to establish whether the cells under study are susceptible to RNAi. - [Read Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes Protocol]

Category: Molecular Imaging: Electron Microscopy Protocols

The protocol for immunolabeling for electron microscopy depends mostly on the primary antibody and its ability to recognize antigen under particular ...Electron Microscopy of the Cytoskeleton of Cultured Cells. Boris Lab. - [Read Electron Microscopy of the Cytoskeleton of Cultured Cells]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

The protocol described in this protocol has been used principally for analyzing the Golgi, endoplasmic reticulum and trans-Golgi network but markers for other compartments (e.g. ERGIC and endosomes) have also been analyzed. Modifications either to the gradient density range or the centrifugation conditions influence the ability of the gradient to resolve multiple compartments. - [Read Fractionation of Golgi, ER, TGN and Other Membrane Compartments in Pre-Formed Iodixanol Gradients]

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: Pharmacology and Toxicology Protocols: Drug Discovery

Protocol describes an assay that relies on the endothelial cells’ ability to form distinct blood-vessel-like tubules in an extracellular matrix  where they can subsequently be visualized by fluorescence microscopy.  Although quantification of the tubules can be performed by manual tracing, this method precludes the use of the assay in unbiased high-throughput applications. - [Read Image Based Assay of Endothelial Cell Tube Formation Protocol]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

Activation and inactivation of proteins using photoactivation of caged peptides or proteins offer insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group. - [Read Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol]

Category: Microscopy Protocols: Confocal Microscopy Protocols

Confocal laser scanning microscopy (CLSM) is a relatively new light microscopical imaging technique which has found wide applications in the biological sciences. The primary value of the CLSM to the biologist is its ability to produce optical sections through a 3-D specimen-e.g., an entire cell or a piece of tissue - that, to a good approximation, contain information from only one focal plane. Article includes principle and applications of confocal laser scanning microscope. - [Read Looking Inside Cells and Tissues by Optical Sectioning with a Confocal Laser Scanning Microscope]

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

Potential embryotoxicity is assessed by monitoring the effect of the test compound on total protein synthesis, and DNA synthesis in cultured human foetal lung fibroblasts.  Rat lung epithelial cells can be used to determine cytotoxicity of select compounds because of their ability to metabolise xenobiotics. - [Read Lung Cell Assay Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for screening immobilized dyes for their ability to bind a target protein. The selection of a dye adsorbent for the purification of the target protein is an empirical undertaking and is generally achieved by a trial-and-error approach. - [Read Screening Immobilized Dyes for their Ability to Bind a Target Protein Protocol]

Category: Stem Cell Protocols: Stem Cell Isolation Protocols

Germ layers are multipotent tissues that have the ability to differentiate into various tissue types. Isolation and manipulation of germ layers is important for examining differentiation. This protocol describes a method for separating postimplantation germ layers. - [Read Separating Postimplantation Germ Layers Protocol]

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

This protocol describes a method for testing of new serum lots prior to use in cell culture. Serum lots vary considerably in their ability to support cell growth, and some lots even contain toxic or growth-inhibitory compounds. It is advantageous to test serum lots and purchase in large volume, both for cost benefit. - [Read Serum Testing for Mammalian Cell Culture Protocol]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Transfection of primary leukocytes has traditionally been a challenging but much desired protocol. It allows not only the analysis of cells in a more natural state to a cell line system, it enables the direct comparison of, for e.g. transcriptional activity using luciferase reporters, in immune cells taken from genetically-altered mice. In addition, importantly it allows for "rescue experiments" in knockout cells & the ability to over-express or reconstitute wild-type and/or mutated constructs. - [Read Transfection of Bone Marrow-Derived Mast Cells for Transcription Factor Luciferase Reporter Assays]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

The principle behind the pressure cooker method described here is to use extended periods of heat to break some of the subcellular structures that block antibody access. This approach is appropriate for handling specimens on glass slides. The major advantages of the pressure cooker method are the ability to handle a large number of slides simultaneously, the convenience of using metal racks, and the avoidance of any hot spots that are found in the microwave. - [Read Unmasking Hidden Epitopes Using the Pressure Cooker Protocol]