procedure Protocols

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Intracellular Ion Measurement Protocols

Protocol for Calcium Flux: Indo-1 loading and sample staining procedure for simultaneous measurement of intracellular Ca2+. Includes: Preparation of INDO-1; Preparation of Ionomycin. - [Read Calcium Flux: Indo-1 Loading and Sample Staining Procedure for Simultaneous Measurement of Intra Ca2]

Category: Transfection Protocols: Calcium Phosphate Transfection Protocols

This protocol,is based on the venerable method of Graham and van der Eb (1973). The procedure is used chiefly to generate stable lines of cells carrying chromosomally integrated copies of the transfected DNA. - [Read Calcium-phosphate-mediated Transfection of Cells with High-molecular-weight Genomic DNA]

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 Cycle Protocols

protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]

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

This protocol describes a method for the synchronization of cell populations using centrifugal elutriation. The method relies on the fact that cell size increases linearly as cells proceed through the cell cycle. Cells of similar size (and cell cycle phase) are eluted stepwise from the cell chamber, with the smallest size (those in early G1) being eluted first. Using this procedure, it is possible to obtain relatively pure populations of cells at various points in G1, S, and G2/M. - [Read Cell Synchronization Using Centrifugal Elutriation Protocol]

Category: Cell Biology and Cell Culture: Cell Viability Protocols

The CellTiter-Blue® Cell Viability Assay uses an optimized reagent containing resazurin. The homogeneous procedure involves adding the reagent directly to cells in culture at a recommended ratio of 20µl of reagent to 100µl of culture medium. - [Read Cell Viability Assays that Measure Metabolic Capacity 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: Signalling Signal Transduction Protocols: Chemokine Protocols

Chemokine Receptor Expression Assay by Flow Cytometry. Mullins Lab. Reagents, Protocol and detailed procedure. - [Read Chemokine Receptor Expression Assay by Flow Cytometry]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Very nice and detailed ChIP assay protocol procedure. Blocking Staph A cells, Crosslinking cells, Chromatin preparation from cell pellets, sonication, blocking. Sugden Laboratory - [Read ChIP ping for Dummies]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Prepare Chromatin, Immunoprecipitate, Wash, Elute, IP Procedure. UpState. - [Read ChIP TIPS PDF]

Category: RNA Protocols: cDNA Libraries Library

Methods and procedure for Colony Hybridization. Vesicle Trafficking Home. - [Read Colony Hybridization Protocol]

Category: ELISA Protocols

Competitive ELISA Protocol. Jill Fergusson, University of Nottingham. - [Read Competitive ELISA Procedure]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

In this protocol, sample and competitor RNAs are reverse transcribed (separately) in a pilot experiment.  A constant amount of sample RT product is then combined with a 2-logserial dilution of competitor RT product for PCR. Procedure provides an approximate copy number for the sample, which is then fine-tuned by repeating the experiment with a series of twofold dilutions of competitor.  The experiment includes controls for sample-to-sample variations in RT efficiency. - [Read Competitive RT-PCR: Estimation of Copy Number Protocol]

Category: Chromatography Protocols: Liquid Chromatography Protocols

Protocol for configuration, column construction, and column packing for a capillary liquid chromatography system. Protocol describes a procedure for adapting conventional HPLC systems to provide accurate low-flow rates (0.4-4 µl/min) and gradients required to operate slurry-packed capillary columns.  A key component of this system is a commercial axial-beam longitudinal flow cell that can be fitted to several commercial UV detectors. - [Read Configuration Column Construction Column Packing for Capillary Liquid Chromatography]

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol is the second step in a three-step process for the preparation and enrichment of phosphopeptides using immobilized metal affinity chromatography (IMAC) for the identification of the phosphopeptides by LCMS/ MS. This procedure describes the construction of microchromatographic columns, or micro-tips. - [Read Construction of Micro-Tip for Use in IMAC Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Cryogenic Preservation and Storage of Animal Cells. PDF.Corning Life Science. Very nice, detailed procedure for the freezing and storage of various cell lines. - [Read Cryogenic Preservation and Storage of Animal Cells - Corning Life Science. PDF.]

Category: Cytogenetics Protocols

Cytogenetic protocol for blood preps. Includes: Preparation of materials; Culture Procedure - Use aseptic technique throughout procedure until harvest; Harvest; Slide making; Collecting blood by tail vein method; PHA Dose Response. - [Read Cytogenetic Protocol Blood Preps]

Category: Signalling Signal Transduction Protocols: Cytokine Protocols

Cytokine Enzyme-linked immunosorbent spot (ELISPOT) assays. Cytokine ELISPOT Buffers and procedure protocol. - [Read Cytokine ELISPOT (Enzyme-linked ImmunoSPOT) Protocols]

Category: RNAi Technology Protocols: RNAi Drosophila Protocols

Protocol describes how subcellular-sized particles are accelerated to high velocity to carry double-stranded RNA (dsRNA) into Drosophila embryos.  The major advantage of this procedure over microinjection (Microinjection of dsRNA into Drosophila Embryos) is that particle bombardment is easier and faster to perform.  In addition, the mechanical trauma received is far less than by microinjection, allowing better survival of embryos and fewer phenotypic artifacts. - [Read Delivery of dsRNA into Drosophila Embryos by Gene Gun Protocol]

Category: Stem Cell Protocols

This protocol decribes derivation of TS cell lines from 3.5-days post coitum (dpc) mouse blastocysts. The procedure is similar to the derivation of embryonic stem (ES) cell lines. However, the success rate is considerably higher, and less expertise is required to recognize pluripotent TS cell colonies. - [Read Derivation of Trophoblast Stem (TS) Cell Lines from Blastocysts Protocol]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Certain fluorescent dyes such as Blankophor have a high affinity for the b -glycosidically linked polysaccharides such as glucan and chitin, which are main the constituents of the fungal cell wall. Therefore, these fluorescent dyes can be used for screening clinical samples for the presence of fungal elements. This procedure can be performed using the following specimens: Nail, Skin, Bronchial alveolar lavage fluid, Sputum and Biopsies. - [Read Detection of Fungi by Fluorescence Microscopy Using Fluorescent Brighteners]

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: Fungi Protocols: Fungi Genetics Protocols

Standard operating procedure for the determination of tissue fungal burden utilizing quantitative real time polymerase chain reaction (QPCR). This standard operating procedure will provide information on how to assess fungal tissue burden of infected animals by use of a single copy (FKS) or multicopy gene (18s RNA) to assess the number of fungal cell nuclei present. - [Read Determination of Tissue Fungal Burden Utilizing Quantitative Real Time Polymerase Chain Reaction]

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

DNA digestion protocol & hints. Procedure and tips for restriction digestion. Doering laboratory, Department Molecular Microbiology, Washington University Medical School. - [Read DNA digestion protocol & hints PDF]