based Protocols

Category: Bioinformatics

Using AFPredictor, it was demonstrated that ‘ordered surface carbons’ (OSCs) are a distinguishing feature of AFPs and, more specifically, their ice-binding surfaces. AFPredictor identified AFPs from within a large set of structures with greater than 99% specificity. Furthermore, it was used to identify a novel ice-binding protein by screening a library of homology modeled structures based on cDNA sequences obtained from cold-acclimated winter rye (Secale cereale). - [Read A Computational Screening protocol for Antifreeze/Ice-Structuring Proteins]

Category: PCR Protocols

The MagneSil system can selectively isolate PCR products that are more than 150-bp long from primers and primer -dimers.  The technology can be used with a number of robotic workstations, including Beckman Coulter’s Biomek 2000 and FX Laboratory Automation Workstations.  The procedure can also be carried out manually.  Typical recovery is more than 80% for a 1-kb product with negligible carryover of primers or nucleotides. - [Read A Magnetic Particle-Based Method for Purifying PCR Products from Solution Protocol]

Category: DNA Protocols: EMSA DNA-Protein Protocols

A non-radioactive electrophoretic mobility shift assay based on the digoxigenin detection system had been developed and applied to the analysis of the interaction between the heat shock transcription factor and the heat shock element of N. crassa. (Bioch - [Read A non-radioactive electrophoretic mobility shift assay for the detection of heat shock element (HSE)]

Category: Molecular Biology Protocols: Carbohydrate Protocols

The method allows the detection and quantification of glycosyltransferase activity using an ELISA-based procedure and carbohydrate-specific monoclonal antibodies. Avoids the use of radiolabeled substrates. Bruce A. Macher~Professor of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA - [Read A Sensitive ELISA-Based Assay for Glycosyltransferases]

Category: Plant Biology Protocols: Plant Genetics Protocols

AFLP was designed as a highly sensitive method for DNA fingerprinting to be used in a variety of fields. We are using this technology to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been identified genetically by mutant phenotype. Protocol includes: Generate polymorphic recombinant F2 (or F3) population; Isolate genomic DNA; Restriction of DNA; Ligation of adapters; Pre-amplification of template DNA; AFLP-PCR; etc. - [Read AFLP For Positional Cloning]

Category: PCR Protocols: AFLP PCR

Ulrich G. Mueller and L. LaReesa Wolfenbarger. Amplified fragment length polymorphisms (AFLPs) are polymerase chain reaction (PCR)-based markers for the rapid screening of genetic diversity. AFLP. TREE October 1999 - [Read AFLP genotyping and fingerprinting Review PDF]

Category: PCR Protocols: AFLP PCR

P Vos, R Hogers, M Bleeker, M Reijans, T van de Lee, M Hornes, A Frijters, J Pot, J Peleman, and M Kuiper. 1995. Nucleic Acid Research. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic - [Read AFLP: a new technique for DNA fingerprinting.]

Category: PCR Protocols: AFLP PCR

Mannie Liscum and Paul Oeller. Department of Plant Biology. Carnegie Institution of Washington, Stanford. AFLP technology is used here to generate DNA based markers for cloning genes involved in phototropic responses in higher plants that have only been i - [Read AFLP: not only for fingerprinting, but for positional cloning]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cytotoxicity Assays in Cell Culture Protocols

Protocol descibes the use of L929 mouse fibroblast cells cultured in vitro in an agarose overlay assay to assess the toxicity of test substances.  The assay may be useful in assessing the irritation potential of test substances (e.g. surfactant-based products) as an alternative to the Draize rabbit eye test. - [Read Agarose Overlay Assay Protocol]

Category: Signalling Signal Transduction Protocols

Protocol on an image-based assay of endothelial cell tube formation as a model of angiogenesis. Includes: Introduction, Methods, Results, Discussion and References. - [Read An Image-Based Assay of Endothelial Cell Tube Formation]

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: PCR Protocols: AFLP PCR

An introduction to AFLP and fAFLP. Mark E. Berres, University of Wisconsin. Amplified fragment-length polymorphism (AFLP) or its fluorescent version (fAFLP) is a PCR-based fingerprinting technology. AFLP basically involves the restriction of genomic DNA - [Read An introduction to AFLP and fAFLP]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

Simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and proteolytic treatment to permeabilize cells. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells for subsequent culturing based on DNA-content differences, is also described. Also presented are methods for staining of cell nuclei isolated from paraffin-embedded tissues, and deconvolution of DNA-content-frequency... - [Read Analysis of Cellular DNA Content by Flow Cytometry Protocol]

Category: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Analysis of DNA Fragmentation Using the JAM Assay. By Shailaja Kasibhatla et al., The JAM assay is based on labeling nuclear DNA of cycling cells with [3H]thymidine and harvesting samples on glass fiber filters. Apoptosis will generate DNA fragments small enough to pass through the glass fiber filter, resulting in decreased radioactivity of the particular sample. Cell-mediated cytotoxicity or cell killing mediated by cytotoxic T lymphocytes (CTL) can also be measured by this technique. - [Read Analysis Of DNA Fragmentation Using The JAM Assay (Subscription Required)]

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

Provides several approaches to flow cytometry data analysis. Frequency determinations based on analysis of single-parameter fluorescence histograms and dual-parameter contour plots are presented. Steps are described for calculating values for signal-to-noise ratios when logarithmic amplification is used for data collection. - [Read Analysis of Flow Cytometry Data Protocol]

Category: Protein Protocols: Protein Trafficking

Protocol is based on methods for the resolution of GLUT4 containing vesicles and the identification of phosphoinositide kinase containing vesicles in 3T3-L1 adipocytes. They may have a wider application to any low-medium density membranes. Protocol incorporates the strategy of using a low density microsome fraction as the gradient input, commonly used in GLUT 4 studies that may have a wider application to other investigations. - [Read Analysis of Membrane Trafficking and Intracellular Signaling in Self-Generated Iodixanol Gradients]

Category: Immunohistochemistry Protocols: Antigen Retrieval Protocols

Protocol used to for immunohistochemistry on paraffin-embedded sections. Based on use of microwave energy to effect antigen retrieval. The immunohistochemistry procedure, is for use of Biomeda's HistoScan kit based on a streptavidin-peroxidase/biotinylated second antibody detection system with 3-amino, 9-ethylcarbazole (AEC) as chromogen. Undoubtedly, other kits or home-made reagents will also work . - [Read Antigen Retrieval for Immunohistochemistry with Paraffin-Embedded Tissues Protocol]

Category: Cell Biology and Cell Culture

Protocol for automated imaging-based high-throughput screening for small molecules that reverse cellular senescence. - [Read Automated Imaging-Based High-Throughput Screening: Small Molecules that Reverse Cellular Senescence]

Category: Bacterial Cell Culture Protocols: Bacterial Stocks Protocols

4 strains of E. coli are used in these studies: JM101 for M13 infection and isolation, XL1BMRF'for M13 or pUC-based DNA transformation, and ED8767 for cosmid DNA transformation. To maintain their respective F' episomes necessary for M13 viral infection, JM101 is streaked onto a M9 minimal media plate and XL1BMRF' is streaked onto an LB plate containing tetracycline. ED8767 is streaked onto an LB plate. These plates are incubated at 37degC overnight. For each strain, 3 ml. of appropriate liquid.. - [Read Bacterial Cell Maintenance Protocol]

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: Transfection Protocols: Calcium Phosphate Transfection Protocols

This calcium phosphate transfection method works best in cell lines that are 1) highly transformed and 2) adherent (Hela, U2OS, SAOS2, AdAH, NPC-KT and obtain from 20% to 100% transfection efficiency depending on the cell line). Works well for transient experiments but precautions should be used in the design and interpretation of experiments based on the discussion below. Also works very well for generating stable cell lines. This method is quite sensitive to the amount of input plasmid. - [Read Calcium Phosphate Transfection Method]

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: Transfection Protocols: Calcium Phosphate Transfection Protocols

Calcium phosphate forms an insoluble precipitate with DNA, which attaches to the cell surface and is taken into the cells by endocytosis. The protocol is easily adapted for use with other types of cells, both adherent and nonadherent. This protocol is a modified version of a method published by Jordan et al. (1996) who rigorously optimized calcium-phosphate-based transfection methods for Chinese hamster ovary cells and the 293 line of human embryonic kidney cells. - [Read Calcium-phosphate-mediated Transfection of Eukaryotic Cells with Plasmid DNAs]

Category: Xenopus Protocols

Protocol describes the use of a basic water-based dye to stain for acid mucosubstances and acetic mucins located on the cartilage of fixed embryos, permitting the examination of cartilage formation. - [Read Cartilage Staining of Xenopus tropicalis Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

This assay is based on carboxyfluorescein labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases. Includes: Working Dilution of FMK-peptide inhibitors; 1X Working Dilution Wash Buffer; Protocol for Flow Cytometry. - [Read CaspaTag Caspase Activity Protocol]