recombinant Protocols

Category: PCR Protocols: Colony PCR

Protocol describes the use of PCR to screen for bacteria that carry recombinant plasmids.  The PCR can be carried out using the same primers as for amplification of the cloned insert.  To determine the orientation of the insert, a third, insert-specific primer that is asymmetrically distanced from the clonal insertion site can be used. - [Read Colony PCR Protocol II]

Category: ELISA Protocols

Detection of Recombinant Proteins by ELISA protocol. Dr. Frank, Oklahoma Medical Research Foundation. - [Read Detection of Recombinant Proteins by ELISA assay]

Category: DNA Protocols: DNA Cloning

Protocols for Recombinant DNA Isolation, Cloning, and Sequencing. Roe and Crabtree. - [Read Protocols for Recombinant DNA Isolation, Cloning, and Sequencing.]

Category: Recombinant Protein Protocols

Isolation of proteins from inclusion bodies Protcol. Expression of recombinant proteins. Dissolving of recombinant protein. Refolding buffer. BJÖRKman group - [Read Isolation of proteins from inclusion bodies]

Category: PCR Protocols: PCR Cloning Protocols

Protocol for blunt-end cloning of PCR products. Incubation of a blunt-end ligation reaction in the presence of an excess amount of an appropriate restriction enzyme can dramatically increase the yield of recombinant plasmids.  The role of the restriction enzyme is to cleave circular and linear concatemers at restriction sites that are re-formed when linear, blunt-ended plasmid molecules ligate to themselves.  I - [Read Blunt-end Cloning of PCR Products Protocol]

Category: Recombinant Protein Protocols: Baculovirus Recombinant Protein

Generation of Recombinant Protein with Baculovirus Infections Protocols and Procedures. Frank Lab. - [Read Insect Cell Culture and Baculovirus Infections]

Category: DNA Protocols: Cosmid Library Protocols

Protocol describes how to construct a library of 35-45-kb fragments of genomic DNA in the double cos site cosmid vector, SuperCos-1. The steps include: Linearization and dephosphorylation of SuperCos-1 DNA; Partial digestion of high-molecular-weight DNA with MboI; Dephosphorylation of high-molecular-weight genomic DNA; Ligation of cosmid arms to genomic DNA: Packaging and plating recombinants; Isolation and analysis of recombinant cosmids: Validation of the library. - [Read Construction of Genomic DNA Libraries in Cosmid Vectors Protocol]

Category: Protein Protocols: Protein Expression Protocols

Baculovirus Production for Recombinant Proteins. Protocol is a modified version of the pharmingen Baculogold kit. Brian Dynlacht. Mike A. Dyer Lab. - [Read Baculovirus Production for Recombinant Proteins]

Category: Immunology: Immunoprecipitation Protocols

Protocols for detection and purification of proteins tagged with a particular epitope, the FLAG tag, although the same general approach can be applied to other epitope tags. The protocols employ the anti-FLAG M2 antibody to detect and purify FLAG-tagged proteins. The methods presented are immunoprecipitation of FLAG fusion proteins from cells using an anti-FLAG M2 affinity gel, detection of FLAG fusion proteins by western blotting, and purification of FLAG fusion proteins by anti-FLAG. - [Read Epitope Tagging of Recombinant Proteins Protocol]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Includes protocols: Direct Adaptation to HyQ© SFX-Insect™ Medium; Sequential Adaptation to HyQ© SFX-Insect™ Medium; Adaptation of High Five™ Cells to HyQ© SFX-Insect™; Production of Recombinant Protein Using the Baculovirus Expression Vector System; Culturing Insect Cells and Production of Baculovirus Expressed Proteins in 2.8 L Fernbach Systems. Production of Autographa californica Multiple Capsid Nuclear Polyhedrosis Virus (rAcMNPV) Stock - [Read Protocols for Insect Cell Culture]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

The use of Ferbach flasks is an ideal method to amplify the cells and virus inoculum for the bioreactor. The method is also very practical for producing sufficient amounts of various proteins for feasibility studies using HyQ© SFX-Insect™ medium. The large surface area in this vessel enables sufficient aeration and, therefore, the cells, when they reach high density, are not oxygen depleted. - [Read Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter]

Category: DNA Protein Interactions Protocols: Electrophoretic Mobility Shift Assays EMSA Protocols

Protocols on band shift. Includes: Phosphorylation of recombinant antibodies; Labeling the antigen with a red fluorophore; Native gel electrophoresis; Band-shift assay for the determination of Kd; Band-shift assay for the determination of koff. - [Read Band Shift Protocols]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins engineered to have a polyhistidine tail at either the carboxyl or amino terminus can easily be purified in one step by affinity chromatography on a resin carrying chelated nickel ions.  Chromatography can be carried out in column or batch formats.  After unbound proteins are washed away, the target protein is eluted using imidazole, which typically preserves the antigenic and functional features of the protein. - [Read Purification of Histidine-tagged Proteins by Immobilized Ni2+ Absorption Chromatography Protocol]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant protein or a chemically synthesized bioactive fragment is immobilized on resin and used as a probe to capture interacting proteins directly from a cell extract.  Affinity-purified proteins are fractionated by gel electrophoresis and visualized by Coomassie staining.  Proteins that interact specifically are identified by comparing this gel profile to one obtained from cell lysates passed over a control resin lacking the immobilized probe protein. - [Read Affinity Purification of Interacting Proteins from Cell Lysates Protocol]

Category: Recombinant Protein Protocols

ELISA detection of preparation of bacterially expressed protein. ELISA REAGENT RECIPES. Frank Lab. - [Read Detection of Recombinant Proteins by ELISA]

Category: General Research Protocols and Methods: Sterile Technique

Sterile technique is one of the most important steps in insuring consistent results when employing recombinant DNA and protein expression techniques. Dr. Chazin Lab, Center for Structural Biology, Vanderbilt Univ. - [Read Sterile Technique for DNA and Protein Expression]

Category: Stem Cell Protocols: Mouse Embryonic Fibroblasts Protocols

This protocol describes the culture of embryonic stem (ES) cells using mitotically inactivating primary mouse embryonic fibroblast (MEF) cells as a feeder layer (preparation described in Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates). The ES culture medium is supplemented with recombinant leukemia inhibitory factor (LIF) to help maintain the cells as pluripotent stem cells. This protocol has been optimized for the ES-D3 cell line. - [Read Propagation of Pluripotent Mouse Embryonic Stem (ES) Cells Protocol]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

A simplified system for rapid generation of recombinant adenoviruses. Includes: Generation of Recombinants in Bacterial Cells; Viral Production in 293 or 911 Cells; Preparation of High Titer Viral Stocks. - [Read A Simplified System for Rapid Generation of Recombinant Adenoviruses]

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins, constructed in pGEX vectors, are fused to glutathione S-transferase (GST) and can be purified to near homogeneity by affinity chromatography on glutathione-agarose.  Bound GST-fusion proteins are readily displaced from the column by elution with buffers containing free glutathione. - [Read Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

A rapid method to analyze the size of the single-stranded DNA of M13 recombinants. - [Read Analysis of Recombinant Bacteriophage M13 Clones Protocol]

Category: Signalling Signal Transduction Protocols: Calcium Measurement Protocols

Shaw et al. Fungal Genetics and Biology 2001. first report of a plant pathogenic fungus for which aequorin-based Ca21 measurement protocols have been developed. - [Read Expression of Recombinant Aequorin as an Intracellular Calcium Reporter PDF]

Category: Stem Cell Protocols: Feeder Cell Preparation Protocols

This protocol describes the culture of embryonic stem (ES) cells using mitotically inactivating primary mouse embryonic fibroblast (MEF) cells as a feeder layer (preparation described in Preparation of Mouse Embryonic Fibroblast (MEF) Feeder Plates). The ES culture medium is supplemented with recombinant leukemia inhibitory factor (LIF) to help maintain the cells as pluripotent stem cells. This protocol has been optimized for the ES-D3 cell line. - [Read Propagation of Pluripotent Mouse Embryonic Stem (ES) Cells Protocol]

Category: Plant Biology Protocols: Arabidopsis Genetics Protocols

Protocols for gene expression and protein localization in Arabidopsis. Includes: Detection of the native protein; Detection of a recombinant version; Immunofluorescence detection in Arabidopsis protoplasts; Isolation of Arabidopsis seedling protoplasts; Subcellular localization of GUS-fusion proteins in Arabidopsis seedlings; Localization of Arabidopsis proteins with GUS in situ enzyme assay. - [Read Gene Expression and Protein Localization in Arabidopsis Protocols]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol for Cytochrome P450. Includes: Microsome Assays General; General Reaction Conditions; Specific Substrates for Cytochrome P450 Isozymes. - [Read Cytochrome P450 Recombinant Enzyme Microsome Protocol]

Category: Cell Culture Protocols: Insect Cell Culture Protocols

Production of recombinant protein using the baculovirus expression vector system (BEVS). - [Read Production of Recombinant Protein Using the Baculovirus Expression Vector System (BEVS)]