choice Protocols

Category: Immunology: Antigen

Antigen Design and Sera Purification. Custom antisera. Sigma Aldrich. Peptide Selection and Design, Coupling Strategy # Selecting the Protein Carrier, Multiple Antigenic Peptides (MAPs), Choice of Host, Adjuvant, Immunization, & Sera Collection, Antisera Purification, Ammonium Sulfate Precipitation, Protein A/G, Immunoaffinity Purification. - [Read Antigen Design and Sera Purification]

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

BN-PAGE has become the method of choice for the investigation of the respiratory protein complexes of the electron transfer chains of a range of organisms. It allows the separation in two dimensions of extremely hydrophobic protein sets for analysis and also provides information on their native interactions. In this review we discuss the capabilities of BN-PAGE in proteomics and the wider investigation of protein:protein interactions with a focus on its use and potential in plant science. - [Read Blue-Native PAGE in Plants: A Tool in Analysis of Protein-Protein Interactions]

Category: C. elegans Protocols: C. elegans Fixing Protocols

Bouin’s fixative is a particularly good choice for worms because it penetrates dense tissues well and is extremely good for fixing antigens. Like all strong fixatives, however, it is unsuitable for some antibody-antigen pairs. In such cases, the length of time in the Bouin’s fixative can be shortened, or paraformaldehyde fixation can be used instead. - [Read Fixing Caenorhabditis elegans in Bouin’s Fixative Protocol]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: DNA Isolation from Mammalian Cells Protocols

Method of choice when large amounts of mammalian DNA are required, for example, for Southern blotting (Rapid Isolation of Mammalian DNA, Rapid Isolation of Yeast DNA, Southern Blotting: Capillary Transfer of DNA to Membranes) or for construction of genomic libraries in bacteriophage {lambda} vectors.  Approximately 200 µg of mammalian DNA, 100-150 kb in length, is obtained from 5 x 107 cultured aneuploid mammalian cells (e.g., HeLa cells). - [Read Isolation of High-molecular-weight DNA from Mammalian Cells Using Proteinase K and Phenol Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

Direct labeling of purified antibodies is the method of choice when simultaneously visualizing two or more antibodies of the same species, class, or subclass. This allows the localization of multiple antigens to be compared in the same cell, tissue, or sample. Labeled primary antibodies are also useful for improving background-to-readout ratios, and they can be essential for immunoassays in which good quantification is needed. - [Read Labeling Antibodies with Fluorochromes Protocol]

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: RNAi Technology Protocols: RNAi Mouse Embryos and Oocytes Protocols

Protocol describes how to introduce a double-stranded RNA (dsRNA) of choice into mouse oocytes or fertilized one-cell embryos by microinjection.  For collection of mouse oocytes and early embryos, see Collection of Mouse Oocytes for RNAi and Collection of Early Mouse Embryos for RNAi. - [Read Microinjection of dsRNA into Mouse Oocytes and Early Embryos Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast PCR Protocols

Information for oligonucleotide details required for PCR. Includes: Primer choice; UPTAG PRIMER; DNTAG PRIMER; UP_45 and DOWN_45 PRIMERS; UP_90 and DOWN_90 PRIMERS. - [Read Oligonucleotide Details for PCR]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the preparation of ion-exchange chromatography column.  Ion-exchange chromatography (IEC) can be used as a crude step in a protein purification scheme, or, with proper preparation, as a high-resolution step.  If high resolution is desired, considerable care should be taken during column preparation, choice of IEC media, and column packing. - [Read Preparation of an Ion-Exchange Column Protocol]

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

Protocol describes an RNA Polymerase III (Pol III) transcription assay using an extract or proteins of choice. Pol III is the polymerase responsible for transcribing 5S RNA, tRNAs, and other small RNAs. α-Amanitin inhibits Pol II transcription in the assay. The newly-transcribed, radiolabeled RNA is visualized by autoradiography following Urea Polyacrylamide gel electrophoresis. - [Read Protocol for Polymerase III In Vitro Transcription]

Category: Microscopy Protocols

Reflected light microscopy is often referred to as incident light, epi-illumination, or metallurgical microscopy, and is the method of choice for fluorescence and for imaging specimens that remain opaque even when ground to a thickness of 30 microns. - [Read Reflected Light Microscopy]

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: Chromatography Protocols: DNA RNA Affinity Chromatography

When many RNA samples are to be processed or when working with small amounts (<50 µg) of total mammalian RNA, the technique of choice is batch chromatography on oligo(dT)-cellulose. The method described in this protocol uses a combination of temperature and ionic strength to maximize binding and recovery of polyadenylated RNA. IMPORTANT: Prepare all reagents used in this protocol with Diethyl pyrocarbonate (DEPC)-treated H2O. Joseph Sambrook and David W. Russell. - [Read Selection of Poly(A)+ RNA by Batch Chromatography - Subscription Required]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

Chromatography on oligo(dT) columns is the preferred method for large-scale purification (>25 µg) of poly(A)+ RNA extracted from mammalian cells. Typically, between 1% and 10% of the RNA applied to the oligo(dT) column is recovered as poly(A)+ RNA. Because the method can be frustratingly slow, it is not recommended for purification of poly(A)+ RNA from multiple samples. For this purpose, batch elution (Selection of Poly(A)+ RNA by Batch Chromatography) is the better choice. - [Read Selection of Poly(A)+ RNA by Oligo(dT)-Cellulose Chromatography - Subscription Required]

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for separating proteins using ion-exchange chromatography. Protocol details the practical considerations of an ion-exchange chromatography (IEC) experiment.  The choice of what type of ion exchanger to use, as well as the composition of the buffers used in this experiment, should be determined prior to beginning this protocol. - [Read Separating Proteins Using Ion-Exchange Chromatography Protocol]

Category: Viral Protocols

Methods and information related to viral detection and concentration in food. Includes: Nucleic acid amplification methods; Recent progress in polymerase chain reaction detection; Detection of human enteric viruses in non-shellfish food; Choice of primers for the detection of human caliciviruses in foods; Biosensors and microarray detection; Alternative confirmation methods. - [Read Viral Detection and Concentration in Food]