determine Protocols

Category: Signalling Signal Transduction Protocols

Protocol allows you to measure the content of cyclic adenosine 3',5'-monophosphate (cyclic AMP or cAMP) in splenic B lymphocytes (B cells) in an enzyme-linked immunoassay. This protocol utilizes acetylation of cAMP to improve sensitivity and reduce interference. Protocol includes information on: how to determine cAMP, calculations and reagents and materials. - [Read Assay of Cyclic AMP in Lysates of Cells]

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

Bradford Protein Assay Spectrophotometry.  Includes spectrophotometry information and the Bradford protein assay: A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution.  A spectrophtometer differs from a colorimeter in the manner in which light is separated into its component wavelengths.  A spectrophotometer uses a prism to separate light and a colorimeter uses filters. - [Read Bradford Protein Assay Spectrophotometry]

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

Includes Abbreviations, Background, and Procedure steps using BSA. The Bradford protein assay (1) is one of several simple methods commonly used to determine the total protein concentration of a sample.  The method is based on the proportional binding of the dye Coomassie to proteins. The assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker.  Coomassie absorbs at 595 nm.  - [Read Bradford Protein Concentration Assay]

Category: Molecular Biology Protocols: Carbohydrate Protocols

o determine the relative amounts of LPS carbohydrates present in a given strain. The assay can be done on one set of samples and then scanned at the various wavelengths for reasonable data on the 3 types of sugars. HEXOSE ASSAY, 6-DEOXYHEXOSE ASSAY, HEPTOSE ASSAY. Hancock Laboratory. - [Read Carbohydrate Assays]

Category: RNA Protocols: cDNA Libraries Library

Protocol for cDNA synthesis and cloning cDNA into plasmid vector. 1st Strand cDNA Synthesis, and determine the efficiency of first strand cDNA synthesis. Also includes second Strand cDNA Synthesis. Dr.Frank - [Read cDNA Synthesis and Cloning]

Category: Protein Protocols: Protein Extraction From Cells

Cell Lysate Extracts. Great protocols for cell lysis preparation from a variety of cell types. There are numerous methods of cell stimulation and lysis. For a given protein, Upstate’s Laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Upstate. - [Read Cell Lysate Extracts]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture. Detection is based on using the luciferase reaction to measure the amount of ATP from viable cells. The amount of ATP in cells correlates with cell viability. - [Read Cell Viability Assays that Measure ATP Protocol]

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: Genetics and Genomics: Cancer Genetics Protocols

Investigators can utilize X chromosome inactivation (methylation) to determine the clonality status of a tumor or premalignant lesion in females. The technique is based on a methylation-sensitive restriction enzyme and analysis of a polymorphic locus on the X chromosome. Clonal cell populations will show "loss" of the non-methylated allele after restriction digest. The assay can be performed on DNA recovered from microdissected samples. Both frozen tissue and fixed-embedded tissue can be used. - [Read Clonality - X Chromosome Inactivation Assay Protocol]

Category: PCR Protocols: Colony PCR

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids.  The PCR can be carried out using the same primers as for amplification of the cloned insert.  To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Most powerful and convincing method to determine if a specific protein is phosphorylated in a physiologically relevant manner is to assay phosphorylation in situ. The procedure described involves incubating cultured cells (e.g., primary neuronal cultures or transfected cells) or tissue preparations (e.g., hippocampal slices) with [32P]orthophosphate, which is then taken up by the cells or tissues and incorporated into the γ-phosphate position of ATP. - [Read Detection of Protein Phosphorylation in Tissues and Cells Protocol]

Category: Plant Biology Protocols: Photosynthesis and Respiration Protocols

This protocol describes how to determine the chlorophyll content of chloroplasts using a spectrophotometric assay. - [Read Determination of Chlorophyll Content in Chloroplasts]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Cell Cycle Analysis Protocols

Describes assays used to determine the distribution of a population of cells to the different stages of the cell cycle as analyzed by flow cytometry. Staining the DNA with different fluorescent dyes, propidium iodide or DAPI, is one of the most direct ways of staging the cells based on DNA content. - [Read Determining Cell Cycle Stages by Flow Cytometry Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

Sequencing conditions tested for the ABI Big-Dye terminators (PE-ABI #4303150 for the 1000 reaction kit - Description: TF,KIT BTD RR-1000) with various templates. Important to quantitate all templates by agarose gel electrophoresis vs size and concentration standards and do a few tests with different template concentrations to determine the optimal conditions for your reactions. Several conditions are given. - [Read Dye Protocols and Notes for Cosmid, BAC, BAC, Fosmid Templates]

Category: PCR Protocols: PCR Optimization

Method for amplifying DNA enzymatically by the polymerase chain reaction (PCR), including procedures to quickly determine conditions for successful amplification of the sequence and primer sets of interest, and to optimize for specificity, sensitivity, and yield.  The first step of PCR simply entails mixing template DNA, two appropriate oligonucleotide primers, Taq or other thermostable DNA polymerases, deoxyribonucleoside triphosphates (dNTPs), and a buffer. - [Read Enzymatic Amplification of DNA by PCR: Standard Procedures and Optimization Protocol]

Category: Antibody Protocols: Epitope Mapping Antibody Protocols

The simplest way to determine whether two monoclonal antibodies bind to distinct sites on a protein antigen is to carry out a competition assay. The assay can be used with antibodies that bind both conformational and linear epitopes, and it is most useful in the analysis of monoclonal antibody specificity because polyclonal sera typically recognize multiple different epitopes. - [Read Epitope Mapping by Competition Assay Protocol]

Category: Signalling Signal Transduction Protocols

This kinase assay is meant to determine whether an agonist or event can influence the autophosphorylation of FAK. The addition of 1 μl of polyGT to the kinase reaction mix will determine the activity of the enzyme against a substrate. Includes information on: Harvest, Immunoprecipitation, Kinase Reaction and Antibody Detection of FAK. - [Read FAK Autophosphorylation Assay]

Category: Developmental Biology: Embryology Protocols

The FAM caspase binding assay kits from ATCC Corporation can be used to determine amounts of active caspases in cells. The FAM-labeled caspase inhibitor can freely diffuse into the cell. Active caspase irreversibly binds the inhibitor. Upon washing the cells, the amount of fluorescence is proportional to the amount of active caspase in the cell. FAM-LETD-fmk (catalog no. 30-1306) is used to detect caspase 8 and FAM-LEHD-fmk (catalog no. 30-1308) is used for caspase 9. - [Read Fam Caspase 8 and 9 Binding Assay for Embryos Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

A crude lysate gel assay can be performed to roughly quantitate the DNA in lysates. This is often a valuable time saving step to determine if the phage yield is sufficient to warrant continuing the procedure. - [Read Gel Assay to Determine DNA Content of Phage Lysates Protocol]

Category: Pharmacology and Toxicology Protocols: Drug Discovery

To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel Elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Lipid Protocols

New screening efforts and chemical modifications of existing compounds have been attempted to identify more selective and potent inhibitors. To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel-elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine gene order using 3-point crosses. - [Read How to Determine Gene Order Using 3-Point Crosses]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine mating type. - [Read How to Determine Mating Type]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine recessive-lethal mutation rates. - [Read How to Determine Recessive-Lethal Mutation Rates]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine the species of a wild-collected isolate. - [Read How to Determine the Species of a Wild-Collected Isolate]