present Protocols

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

The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. - [Read A Novel Fluorescent pH Probe for Expression in Plants]

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: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols

Protocol for CellTiter-Glo luminescent cell viability assay. This assay is a homogeneous method of determining the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells. - [Read CellTiter-Glo Luminescent Cell Viability Assay Protocol]

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Immobilized metal ion affinity chromatography (IMAC) exploits a molecule’s affinity for chelated metal ions.  The amino acid histidine present in many proteins forms complexes with transition metal ions such as Cu2+, Zn2+, Ni2+ and Fe3+.  Chelating Sepharose™ Fast Flow with a suitable immobilized metal ion will therefore selectively retain proteins with exposed histidine. - [Read Chelating Sepharose Fast Flow 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: PCR Protocols: In-situ PCR Protocols

In Situ Polymerase Chain Reaction: Past, Present, Future. Roche. - [Read In Situ Polymerase Chain Reaction Roche]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

GFP serves as a molecular marker that can be imaged dynamically in living cells, both in its native form & as a fusion to other proteins. For GFP imaging, plants present the challenge of autofluorescence from chlorophyll, lignified cell walls, vacuolar contents, and other cell materials, all of which can obscure the GFP signal. Maximizing the signal-to-noise ratio is a major concern, and careful consideration should be given to the choice of tissue imaged, GFP expression level, etc. - [Read Live-Cell Imaging of GFP in Plants]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: DNA Microarray Protocols: Troubleshooting DNA Microarray

Images present some common problems encountered during microarray production, with possible solutions. Galbraith laboratory. - [Read Microarray troubleshooting: image gallery]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Primer pairs will amplify sequences present as a single copy in the mouse genome with the Universal Genotyping Protocol. Includes: b-Galactosidase (LacZ); cre-recombinase; CFP; diphtheria toxin; dsRED; Fabpi-200; Fabpi-500; flp recombinase; GFP/BFP/YFP; human growth hormone (complete); human growth hormone (transcriptional stop); luciferase (click-beetle); luciferase (firefly); neomycin phosphotransferase; SRY (male-specific); tTA (tet-on). - [Read PCR Genotyping Primer Pairs Protocols]

Category: Pharmacology and Toxicology Protocols: Pharmacokinetics Protocols

The purpose of the present protocol is to obtain this information through collaboration with interested physicians and patients taking part in the European AED Pregnancy Registry (EURAP). - [Read Pharmacokinetic Evaluation of Antiepileptic Drugs During Pregnancy]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Plasmodium vivax is the second most prevalent malaria parasite affecting more than 75 million people each year, mostly in South America and Asia. In addition to major morbidity this parasite is associated with relapses and a reduction in birthweight. Practical PCR genotyping protocols based on polymorphic loci present in two P. vivax genetic markers. - [Read Practical PCR Genotyping Protocols for Plasmodium vivax using Pvcs and Pvmsp1]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

A procedure for direct and indirect staining of single-cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single-cell suspensions is also included. - [Read Preparation of Cells and Reagents for Flow Cytometry Protocols]

Category: Immunology: B Cell Protocols

Describes procedures for measuring the capacity of purified B cells to undergo proliferation. The method centers on the use of polyclonal stimulating agents (mitogens) because these agents stimulate the majority of B cells and because the alternative (measurement of antigen-induced proliferation) requires the laborious procedures of isolating antigen-specific B cells (which are otherwise present in too low a concentration in whole B cell populations). - [Read Proliferative Assays for B Cell Function Protocol]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

This protocol uses a "light mitochondrial" pellet from a mammalian liver homogenate. The gradient thus has to resolve a variety of denser components (peroxisomes, lysosomes, mitochondria) from the Golgi membranes, which have a low density in iodixanol (1.06-1.09 g/ml) [1]. The protocol is specifically tailored to the purification of Golgi membranes from this pellet and is unsuitable for the isolation or analysis of other organelles present in the light mitochondrial fraction. - [Read Purification of Golgi Membranes from a Light Mitochondrial Fraction in a Self-Generated Gradient]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Peroxisome Isolation Protocols

Peroxisomes can be purified in self-generated iodixanol gradients in high yield (80-90%) with no detectable contamination from any other organelle. In iodixanol peroxisomes are the densest of the major subcellular organelles (ρ = 1.18-1.20 g/ml) present in the light mitochondrial fraction from mammalian tissues and cells. - [Read Purification of Peroxisomes in a Self-Generated Gradient]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for selection of an ion exchanger: Determining the pI of a protein using isoelectric focusing. The choice of whether to use an anion or a cation exchanger should be based on knowledge of the stability of the protein, and the binding properties of the target protein and other molecules present. - [Read Selection of an Ion Exchanger Protocol]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for selection of an ion exchanger: Determining the pI of a protein using the titration curve method. The choice of whether to use an anion or a cation exchanger should be based on knowledge of the stability of the protein, and the binding properties of the target protein and other molecules present in the sample. - [Read Selection of an Ion Exchanger Protocol II]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for selection of an ion exchanger: Determining the pI of a protein using the test tube method. The choice of whether to use an anion or a cation exchanger should be based on knowledge of the stability of the protein, and the binding properties of the target protein and other molecules present in the sample. - [Read Selection of an Ion Exchanger Protocol III]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for selection of an ion exchanger: Determining the pI of a protein using the Trial-and-Error method. The choice of whether to use an anion or a cation exchanger should be based on knowledge of the stability of the protein, and the binding properties of the target protein and other molecules present in the sample. - [Read Selection of an Ion Exchanger Protocol IV]

Category: Plant Biology Protocols: Arabidopsis Transformation Protocols

Protocol for a simplified Arabidopsis transformation. Found that the MS salts, hormone, etc. make no difference, that OD of bacteria doesn't make much of a difference, that vacuum doesn't even make much of a difference as long as you have a decent amount of surfactant present. Plant health is still a major factor - healthy fecund plants make a big difference! With this method you should be able to achieve transformation rates above 1%. - [Read Simple Arabidopsis Transformation Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for southern blotting: simultaneous transfer of DNA from a single agarose gel to two membranes. DNA can be simultaneously transferred from opposite sides of a single agarose gel to two membranes.  Bidirectional transfer occurs rapidly at first, but soon slows down as the gel becomes dehydrated.  Because the efficiency of transfer is low, the method works best when the target sequences are present in high concentration - [Read Southern Blotting: Simultaneous Transfer of DNA from a Single Agarose Gel to Two Membranes Protocol]