molecule Protocols

Category: Molecular Biology Protocols: Carbohydrate Protocols

The ABH crosslinker binds to the Fc portion of an antibody molecule, away from the antigen binding site, resulting in a divalent immunologically active immunoglobulin derivative. Pierce - [Read ABH (p-Azidobenzoyl hydrazide) PDF]

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: Immunology: Chemotaxis

Chemotaxis Assay, Springer Lab. A chemotaxis assay's function is to assess whether a factor or molecule of interest has chemotactic activity on a motile cell type. Chemotaxis is the ability of a factor to cause the migration of a cell. The chemotactic assay is based on the creation of a chemical gradient of the chemotactic agent which will cause cells to migrate through the gradient towards the chemotactic agent. - [Read Chemotaxis Assay]

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: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

The first step in competitive RT-PCR is the synthesis and purification of the synthetic competitor.  This is an RNA molecule designed to be reverse-transcribed and PCR-amplified with the same efficiency as the endogenous transcript of interest.  Once the competitor molecule has been prepared, as described in this protocol, competitive PCR can be carried out. - [Read Competitive RT-PCR: Preparation of Competitor RNA Protocol]

Category: C. elegans Protocols: C. elegans Cell Culture Protocols

Protocol for Dauer pheromone prep. Includes small molecule prep and cleaner prep. - [Read Dauer Pheromone Prep Protocol]

Category: Microscopy Protocols: Live-Cell Imaging Protocols

When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected. Includes synthesis of caged peptides or proteins. - [Read Design, Synthesis, and Characterization of Caged Compounds]

Category: Microinjection Protocols: Microinjection of Proteins Protocols

When choosing a particular molecule for photoactivation studies, it is necessary to have some structural knowledge of the molecule in order to design an appropriately caged species that will retain its biological inactivity until uncaging is effected. - [Read Design, Synthesis, and Characterization of Caged Compounds Protocol]

Category: Genetics and Genomics: Genotyping Protocols: PCR Genotyping

Developed PCR-based single molecule haplotyping methods that enable both surveys for novel inversion variants, and population-scale genotyping of known inversions - [Read Haplotype-Fusion PCR Protocol]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Provides a protocol for indirect immunofluorescence, which is a method that provides information about the locations of specific molecules and the structure of the cell. Antibody molecules for a specific target molecule are exposed to the cell or tissue being investigated. The binding of these molecules is detected by incubating the sample with a secondary antibody specific for immunoglobulin molecules and conjugated to fluorophore. - [Read Immunofluorescence Staining Protocol]

Category: Microscopy Protocols: Fluorescence Microscopy Protocols

Multiphoton fluorescence microscopy is a powerful new technology that enables the acquisition of optical sections without the use of a pinhole aperture typically used for confocal microscopy. The technique is based upon the two-photon principle: A fluorescent molecule simultaneously absorbs two photons producing an electronic transition from the ground to excited state equal to two times the energy of each incident photon. - [Read Multiphoton Images from LSM 510 NLO System]

Category: Avian Bird Protocols

Protocol describes the production of double-stranded RNA (dsRNA) from fragments of cDNAs of candidate genes. The cDNA fragments must be cloned in plasmids with a flanking SP6 and T7 promoter (e.g., pSP72 or pCRII). The plasmid is linearized and sense and antisense RNAs are produced separately by in vitro transcription. After purification, the RNA strands are annealed to yield a dsRNA molecule suitable for RNAi in avian embryos. - [Read Production of dsRNA for RNAi in Avian Embryos Protocol]

Category: Cell Biology and Cell Culture

This protocol focuses on the interactions between L-selectin expressed on neutrophils and PNAd coated onto the plastic surface. The main purpose of the flow chamber assay is to visualize and measure interactions between flowing cells expressing a given adhesion molecule on their surface, and their receptor, either directly coated on the flow chamber lower wall or expressed on a cell monolayer. - [Read Protocol for L-selectin-PNAd Interactions under Flow Conditions.]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

This protocol describes a method for reverse transcriptase (RT) in situ PCR. In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step. The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in RNase-free DNase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

Category: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol describes a method for reverse transcriptase (RT) in situ PCR.  In situ PCR differs from PCR in situ hybridization in the inclusion of a reporter molecule in the amplification step.  The two steps of RT in situ PCR that differ from in situ PCR are overnight digestion in Rnase-free Dnase that is performed after protease digestion, and an RT step, prior to in situ PCR. - [Read Reverse Transcriptase In Situ PCR Protocol]

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

Small ubiquitin-like modifier (SUMO) is a small molecule, but has a variety of regulatory functions in cells. SUMO modification is involved in transcriptional regulation, subcellular localization, and protein-protein interactions. SUMO conjugation requires sequential E1-dependent activation, E2-dependent conjugation, and E3-dependent ligation steps. Protocol includes: In vivo and in vitro SUMOylation assay and deSUMOylation assay. - [Read Sumoylation and Desumoylation Assays for a Chromatin-Remodelling Complex In Vivo and In Vitro]