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A Novel Fluorescent pH Probe for Expression in Plants - http://www.plantmethods.com/content/2/1/7

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]

A Simplified System for Rapid Generation of Recombinant Adenoviruses - http://www.coloncancer.org/adeasy/protocol.htm

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]

Affinity Purification of Interacting Proteins from Cell Lysates Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4593

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]

AFLP For Positional Cloning - http://www-ciwdpb.stanford.edu/publications/methods/aflp.html

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]

Amplification and Storage of a Cosmid Library: Amplification on Filters Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4002

Category: DNA Protocols: Cosmid Library Protocols

Amplification of cosmid libraries may result in distorted representation of cloned genomic sequences and should be avoided wherever possible. In this method of amplification, distortion of the library is rarely a problem because at no stage are bacteria containing different recombinant cosmids grown in competition with one another. - [Read Amplification and Storage of a Cosmid Library: Amplification on Filters Protocol]

Analysis of Recombinant Bacteriophage M13 Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3996

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]

Baculovirus Production for Recombinant Proteins - http://axon.med.harvard.edu/~cepko/protocol/mike/B1.html

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]

Band Shift Protocols - http://www.pharma.ethz.ch/institute_groups/biomacromolecules/protocols/bandshift

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]

Blunt-end Cloning of PCR Products Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3830

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]

Colony PCR Protocol II - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4141

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]

Construction of Genomic DNA Libraries in Cosmid Vectors Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4000

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]

Culturing of Insect Cells and Production of Baculovirus Expressed Recombinant Proteins in 2.8 Liter - http://www.hyclone.com/pdf/procedure_insect_fernbach.pdf

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]

Cytochrome P450 Recombinant Enzyme Microsome Protocol - http://www.researchd.com/cyp450/microsom.htm

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]

Detection of Recombinant Proteins by ELISA - http://omrf.ouhsc.edu/~frank/ELISA.html

Category: Recombinant Protein Protocols

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

Detection of Recombinant Proteins by ELISA assay - http://omrf.ouhsc.edu/~frank/ELISA.html

Category: ELISA Protocols

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

Epitope Tagging of Recombinant Proteins Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E663998FCCB61070F32F1EAE0301F7F&objectid=6677C749FA75FD435618AC07256968A7

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]

Expression of Recombinant Aequorin as an Intracellular Calcium Reporter PDF - http://www.biology.ed.ac.uk/research/institutes/plant/PDF/2001/Shaw-2001-207.pdf

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]

Gene Expression and Protein Localization in Arabidopsis Protocols - http://www.arabidopsis.org/cshl-course/7-gene_expression.html

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]

Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4078

Category: Genetics and Genomics: Genotyping Protocols: Mutation Detection Protocols

The double-stranded DNA of recombinant plasmid, phagemid, or bacteriophage M13 replicative form DNA is digested with two restriction enzymes whose sites of cleavage both lie between one end of the target DNA and the binding site for universal primer. The enzyme that cleaves nearer the target sequence must generate either a blunt end or a recessed 3' terminus; the other enzyme must generate a four-nucleotide protruding 3' terminus. - [Read Generation of Sets of Nested Deletion Mutants with Exonuclease III Protocol]

Growth of S. cerevisiae and Preparation of DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3298

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes methods for isolation of DNA from a strain of S. cerevisiae carrying a recombinant YAC. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. The method is suitable for preparing DNA that will be used for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high-molecular-weight DNA. - [Read Growth of S. cerevisiae and Preparation of DNA Protocol]

Harvesting the mRNA for Exon Trapping and Amplification - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3734

Category: DNA Protocols: Genomic DNA Library Protocols

Standard techniques are used here to isolate cytoplasmic RNA from COS cells transfected with a library of recombinant plasmids containing the genomic DNA of interest. - [Read Harvesting the mRNA for Exon Trapping and Amplification]

Hybridization of Bacteriophage DNA on Filters Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3983

Category: Viral Manipulation Protocols: Phage Protocols

Using hybridization, it is possible to identify a single recombinant that carries the desired target sequence on a filter that carries the imprint of 15,000 or more plaques. - [Read Hybridization of Bacteriophage DNA on Filters Protocol]

Insect Cell Culture and Baculovirus Infections - http://omrf.ouhsc.edu/~frank/BACULPRO.html

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]

Isolation of proteins from inclusion bodies - http://www.its.caltech.edu/%7Ebjorker/Isolation%20of%20proteins%20from%20.pdf

Category: Recombinant Protein Protocols

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

K. lactis Protein Expression Kit PDF - http://www.neb.com/nebecomm/ManualFiles/manualE1000.pdf

Category: Protein Protocols: Protein Expression Protocols

K. lactis Protein Expression Kit. Recombinant Protein Expression in Yeast. NEB Biolabs - [Read K. lactis Protein Expression Kit PDF]

Articles (1-2 of 2)

DNA Ligation Protocol

The DNA Ligation protocol described here contains the steps required to join together using ligase enzyme both plasmid DNA and insert DNA fragments in order to create a new plasmid. This new ligated plasmid can be transformed after into competent bacteria to produce DNA for mini, midi or maxi-prep isolation.

Vector Design Protocol for Transgenic Mice Generation

The protocol gives general considerations for the design of targeting vectors for transgenic mice. The protocol shares tips in the design of knock-out and knock-in vectors and some of their strategies for producing homologously recombined embryonic stem cells.

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