functional Protocols

Category: RNA Protocols: 5' RACE - Rapid Amplification of cDNA Ends Protocols

Can also use 96-well plates. First Strand cDNA Synthesis, First Size selection, second 2nd strand cDNA synthesis. Bay Area Functional Genomics Consortium, UCSF. - [Read 5' RACE Protocol For Generation of Seq. Tags]

Category: Fungi Protocols: Aspergillus Protocols

The technique makes use of an Escherichia coli strain expressing the redΑßΓ operon under the control of an inducible promoter. This enables the strain to carry out homologous recombination with only 50-60 bp of homologous sequence. The procedure does not require any DNA ligation and is very rapid. It allows a single gene or region on a cosmid to be replaced by a bi-functional selectable marker (having both an E. coli and an A. fumigatus marker). - [Read A Rapid Method for Generating Gene Deletions in Aspergillus fumigatus Protocol]

Category: Neuroscience Protocols

After section of the corticospinal (CS) tract in adult rats, neutralizing Nogo-A with monoclonal antibodies leads to enhancement of axonal re-growth and compensatory sprouting, accompanied by increased motor recovery.  The neutralization of Nogo-A represents a promising approach for therapy after lesion if its enhancement of functional recovery can be transposed to primates. - [Read Anti-Nogo-A Treatment Protocol]

Category: Reporter Gene Assays: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-gal gene. If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-gal gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta-gal will work as indicator host bacteria; a single chromosomal copy of the gene is not a problem. - [Read Assay for Phage Containing the Beta-galactosidase Gene]

Category: Yeast Protocols: Yeast Genetics Protocols: B-Galactosidase Assay Protocols

This assay is used when working with phage vectors carrying the beta-galactosidase gene (often used for immunological screening). If the cloning event disrupts a normally functional copy of the gene in the vector the resulting plaques would appear clear in the assay. If the phages contain a functional beta-galactosidase gene they will form blue rings around their plaques. Any strain which is not an overproducer of beta- galactosidase will work as indicator host bacteria. - [Read Assay for Phage Containing the Beta-galactosidase Gene Protocol]

Category: Neuroscience Protocols: Neuronal Cell Culture Protocols: Rat Neuronal Cell Culture Protocols

Conditional ablation of stat3/socs3 discloses the dual role for reactive astrocytes after spinal cord injury. The current protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). - [Read Conditional Ablation of Stat3 Socs3 Discloses the Dual Role for Reactive Astrocytes]

Category: Cell Biology and Cell Culture: Cell Cycle Protocols: Mitosis Protocols

Cytostatic factor extract (CSF) preparation for spindle assembly protocol. Protocol includes tips such as: The quality of the eggs is essential for good CSF extracts. Always sacrifice quantity for quality when trying to make functional extracts. - [Read Cytostatic Factor (CSF) Extract Preparation for Spindle Assembly Protocol]

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol for cytostatic factor extract preparation for spindle assembly. The quality of the eggs is essential for good CSF extracts. Always sacrifice quantity for quality when trying to make functional extracts. Discard any batches of eggs that have "puff balls" or activated eggs that constitute more than 10% of the eggs. Use laid eggs and collect eggs at about 16 to 17 hours after priming with Progesterone (found in Pregnant Mare Serum Gonadotropin (PMSG)). - [Read Cytostatic Factor (CSF) Extract Preparation for Xenopus Spindle Assembly Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

Acidocalcisomes, the dense acidic calcium-storing organelles, which were originally identified in Trypanosoma cruzi, have no parallels in mammalian cells. They thus represent a unique functional characteristic, not shared by the host and hence offer an important potential target for chemotherapy of Chagas disease. - [Read Fractionation of Acidocalcisomes and Other Organelles from Trypanosoma, Leishmania, Chlamydomonas]

Category: Antibody Protocols: Antibody Production Protocols

Novel strategy of immunizing a phosphorylated peptide or a synthetic phosphopeptide, which corresponds to the protein phosphorylated at a targeted residue. Method has been applied to the production of antibodies that can specifically recognize the other types of site-specific protein modification, such as acetylation, methylation, and proteolysis. - [Read Functional Analyses for Site-Specific Phosphorylation of a Target Protein in Cells]

Category: Microscopy Protocols: In Vivo Imaging Protocols

Positron emission tomography (PET) is a established quantitative and noninvasive imaging modality. With the PET reporter gene (PRG)/PET reporter probe (PRP) system, based on a mutant form of herpes simplex virus 1 thymidine kinase (HSV1-sr39tk), the PET signal is directly proportional to the enzymatic activity of sr39TK9-14. In this protocol, we describe in detail a method for reporter gene labeling of islets and quantitative scanning using a reporter probe. - [Read In Vivo Functional Real-Time Imaging of Transplanted Islets Using Positron Emission Tomography (PET)]

Category: Transfection Protocols: Transient Transfection Protocols

Transient transfection of genes into naïve mouse T cells using nucleofection using a modified electroporation technique. This protocol offers the possibility for rapid functional in vivo studies of targeted genes in primary mouse T cells. - [Read Nucleofection and Adoptive Transfer of Primary Mouse T lymphocytes using Transient Transfection]

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: Signalling Signal Transduction Protocols

Protocol demonstrates that reactive astrocytes play a crucial role in wound healing and functional recovery by using mice with a selective deletion of the signal transducer and activator of transcription-3 (STAT3) or suppression of cytokine signaling-3 (SOCS3) under the control of Nestin gene promoter/enhancer (STAT3N–/–, SOCS3N–/–). Procedure includes: Surgical procedures, Functional evaluation, Immunohistochemistry, In vitro migration assay. - [Read Protocol for Conditional Ablation of stat3/socs3 Discloses the Dual Role for Reactive Astrocytes]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins engineered to have a polyhistidine tail at either the carboxyl or amino terminus can easily be purified in one step by affinity chromatography on a resin carrying chelated nickel ions.  Chromatography can be carried out in column or batch formats.  After unbound proteins are washed away, the target protein is eluted using imidazole, which typically preserves the antigenic and functional features of the protein. - [Read Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography Protocol]

Category: Immunology: T Cell Protocols

In the first protocol, IL-2-producing murine T cells are measured following stimulation by the mitogen Con A. The second protocol provides a modification for using human responder cells. The second protocol is used for estimating the proportion of cells that can generate a clone of cytotoxic effector cells when stimulated by Con A with the addition of IL-2. - [Read Quantitation of Functional T Cells by Limiting Dilution Protocols]

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

In this test rabbit articular chondrocytes are cultured in the presence of test compound, the toxicity of which is then determined by its effect on the production of proteoglycan by the cells, as detected by the dye Alcian Blue. - [Read Rabbit Articular Chondrocyte Functional Toxicity Test]

Category: DNA Protocols: Genomic DNA Library Protocols

Amplified cDNAs containing functional 3' and 5' splice sites are selected by digestion with BstXI and cloned into a Bluescript vector. - [Read Reverse Transcriptase-PCR for Exon Trapping and Amplification]

Category: Reporter Gene Assays: Luciferase Assay Protocols

Protocol describes a split luciferase complementation assay used to study the interaction of proteins in cells. In the split protein strategy, a single reporter protein/enzyme (firefly luciferase [Fluc]) is cleaved into amino-terminal and carboxy-terminal halves; each half is fused to one of two interacting proteins, X & Y. Physical interactions between the two proteins reconstitute the functional reporter protein, leading to enzymatic activities that can be measured by in vitro or in vivo assay - [Read Split Luciferase Complementation Assay for Studying Interaction of Proteins X and Y in Cells]

Category: E Coli Protocols: E coli Genetics Protocols

Protocol for systematic mutagenesis of E.coli K-12 MG1655 ORFs. Includes: Mutagenesis Project Progress; Construction of Sce-poson mutants; Mutagenesis of Essential Genes; Gene Gorging. - [Read Systematic Mutagenesis of E.coli K-12 MG1655 ORFs Protocol]

Category: Cloning Protocols: Mutagenesis Protocols

Protocol for systematic mutagenesis of e. coli K-12 MG 1655. - [Read Systematic Mutagenesis of E.coli K-12 MG1655 ORFs Protocol]