activity Protocols

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

Using excitation at 365 nm and measuring emission at 455 nm, the amount of 4-MU produced can be quantified. Under these conditions, background fluorescence from the substrate is negligible, especially if the appropriate filter is selected. - [Read Quantitative GUS Activity Assay of Plant Extracts]

Category: Nucleic Acid Modification Protocols: Nucleic Acid Radiolabeling Protocols

In this procedure, synthesis of cDNA is carried out in the presence of saturating concentrations of all four dNTPs and trace amounts of a single radiolabeled dNTP.  After subtraction hybridization, the enriched single-stranded cDNA is radiolabeled to high specific activity in a second synthetic reaction by extension of random oligonucleotide primers using the Klenow fragment of E. coli DNA polymerase. - [Read Radiolabeling of Subtracted cDNA Probes by Random Oligonucleotide Extension Protocol]

Category: Signalling Signal Transduction Protocols

CHIESA et al 2001. J Biochem. Calcium-sensitive photoprotein aequorin used to analyse Ca2+ homoeostasis at the subcellular level. GFP chimaeras for studying the recruitment of protein kinase C isoforms and the activity of intracellular proteases. - [Read Recombinant aequorin and green fluorescent protein as valuable tools in the study of cell signalling]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

How much DNA to digest? Master mix for restriction digest. Double digests. Star activity. Matt Lewis, Department of Pathology, Univ. Liverpool. - [Read Restriction enzyme digestion of DNA: basic method]

Category: RNA Protocols: RT-PCR and cDNA synthesis

This procedure was first described by Bertrand et al to demonstrate that ribozymes could be enzymatically active in vivo. We adapted the method to show that certain oligodeoxynucleotides could direct the activity of endogenous ribonuclease H to cleave tar - [Read Reverse Ligation Mediated RT - PCR]

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

In vitro transcription systems includes instructions for use of products P1420, P1430, P1440, P1450 and P1460.Includes information and protocols on RNA Transcription in vitro. Information on DNA Template Preparation;Synthesis of High-Specific-Activity Radio labeled RNA Probes;Determining Percent Incorporation and Probe Specific Activity;Removal of the DNA Template Following Transcription;Removal of unincorporated nucleotides;Synthesis of large amounts of RNA;Capping RNA for in vitro translation. - [Read Riboprobe In Vitro Transcription Systems]

Category: Genetics and Genomics: Cancer Genetics Protocols

SAGE is a new method that has been invented at Johns Hopkins University in USA to give scientists an overview of a cell’s complete gene activity. It works by capturing RNAs, identifying them and counting them. By comparing different types of cells, the researchers hope to generate profiles that will help them understand healthy cells and what goes wrong during diseases. Includes: How SAGE works and Steps of SAGE. - [Read Serial Analysis Of Gene Expression (SAGE)]

Category: Cell Biology and Cell Culture

Protocol for telomerase PCR ELISA. Fast and sensitive detection of telomerase activity. - [Read Telomerase PCR ELISA Protocol]

Category: ELISA Protocols

Protocol for telomerase PCR ELISA. For fast and sensitive detection of telomerase activity. - [Read Telomerase PCR ELISA 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: Yeast Protocols

Plasma membranes are isolated from the yeast Saccharomyces cerevisiae. The cell wall is initially digested by helicase, followed by hypoosmotic lysis and homogenization. Membranes are prepared by subsequent differential centrifugation. The activity of the H+-ATPase is then determined by measuring the amount of inorganic phosphate released from ATP. - [Read Yeast Plasma Membrane H+ -ATPASE Toxcity Test Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Microorganism Cytotoxicity Assays Protocols

Accumulation of lipophilic substances, many of which may be environmental chemicals, affects the membrane lipid order and consequently affects the functions of these proteins.  Since, the function of important cellular proteins, such as the H+-ATPase strongly depends upon the integrity of the lipid bilayer, the activity of the H+-ATPase may be used as a sensitive indicator of the effect that a chemical may have on the viability of the cell. - [Read Yeast Plasma Membrane H+ -ATPASE Toxicity Test]