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Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3597

Category: E Coli Protocols: E coli Protein Protocols

Protocol for the expression of cloned genes in E. coli using the bacteriophage T7 promoter. Protocol describes how (1) to clone cloned sequences encoding open reading frames in plasmids carrying bacteriophage T7 promoters, (2) to optimize expression of target proteins in transformants carrying these recombinants, and (3) to scale-up production of foreign proteins. - [Read Expression of Cloned Genes in E. coli Using the Bacteriophage T7 Promoter Protocol]

Expression of F-tryptophan labelled protein - http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/fluoexp.html

Category: Protein Protocols: Protein Expression Protocols

Expression of F-tryptophan labelled protein. Protocol can be adapted for Fluoro-phenylalanine or Fluro-tyrosine labelling. Transformation of competent E. coli cells for expression with plasmid. Dr. Chen, Dept. of Biochem. & Mol. Biology, University College, London. - [Read Expression of F-tryptophan labelled protein]

Expression of protein in minimal media - http://www.biochem.ucl.ac.uk/bsm/nmr/protocols/protocols/protexp.html

Category: Protein Protocols: Protein Expression Protocols

Expression of protein in minimal media using E. coli cells for expression. Dr. Chen, Biochem. & Mol. Biology, University College. London - [Read Expression of protein in minimal media]

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]

Expression Protocol in M9 Minimal Media via T7 Promoter - http://structbio.vanderbilt.edu/chazin/wisdom/labpro/M9.html

Category: Microbiology: Bacterial E.Coli Culture Media & Plates

How to prepare M9 minimal media and grow in M9 media. Chazin Lab , Vanderbilt Univ. - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

Expression Protocol in M9 Minimal Media via T7 Promoter - http://structbio.vanderbilt.edu/chazin/wisdom/labpro/M9.html

Category: Bacterial Cell Culture Protocols: Bacterial Cell Culture Media Protocols

This protocol has been used successfully to 15N or 13C/15N label our proteins using our pET1120/BL21(DE3) expression system: Preparing M9 minimal media begins with preparing a 5x stock solution of M9 salts. Generally, M9 salts contain a nitrogen source in the form of NH4Cl. Since we want to add a labeled nitrogen source, our 5x salts are prepared minus NH4Cl. Standard 5 X M9 Minimal Media salts minus nitrogen source For 1L 5xM9 salts: - [Read Expression Protocol in M9 Minimal Media via T7 Promoter]

First strand cDNA synthesis with Super Script II Protocol - http://www.methods.info/Methods/cDNA/SSII.html

Category: PCR Protocols: cDNA Synthesis Protocols

This method of first strand cDNA synthesis is more efficient than Ready-to-Go beads. You can use as little as 100ng total RNA and still detect you gene expression in PCR. - [Read First strand cDNA synthesis with Super Script II Protocol]

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]

Genome-Wide Expression Protocol - http://web.wi.mit.edu/young/expression/tech.html

Category: Yeast Protocols: Yeast Genetics Protocols

Protocol for genome-wide expression. - [Read Genome-Wide Expression Protocol]

Growth of Primary Cell Culture and Viral Vector Handling Protocols - http://www.stanford.edu/dept/EHS/prod/researchlab/bio/docs/Palmer_Bio_protocol.pdf

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Growth of Primary Cell Culture and Viral Vector Handling Protocols]

Identification of Positive GATEWAY Expression Clones Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.prot4647

Category: Bacteria Genetics Protocols

Protocol for the identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection. Protocol describes ways in which difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances. - [Read Identification of Positive GATEWAY Expression Clones Protocol]

Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3785

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol - http://www.cshprotocols.org/cgi/content/extract/2007/3/pdb.ip28

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.prot4603

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Using confocal laser-scanning microscope & GFP fusion proteins in time-lapse imaging to visualize the behavior of organelles and to track membrane-bound transport intermediates that bud off from organelles. Practical issues related to construction & expression of GFP fusion proteins are discussed. Essential for optimizing the brightness and expression levels of GFP fusion proteins so that intracellular membrane-bound structures containing these fusion proteins can be readily visualized. - [Read Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells]

Immunofluorescent Staining for the Laser Microdissection of Individual Cells Protocol - http://www.natureprotocols.com/2006/08/03/immunofluorescent_staining_for.php

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

Useful techniques to circumvent disruption of tissue structure in the analysis of gene expression are LCM and LDM. While they require specialized microscopes and systems, they are similar in that freshly-cut frozen tissue sections can be microdissected using either a general histological stain (like H&E) or by staining with fluorescently conjugated antibodies. The LCM system by Arcturus involves... - [Read Immunofluorescent Staining for the Laser Microdissection of Individual Cells Protocol]

Isolation and Growth of Primary Cell Cultures from Mouse Protocol - http://www.stanford.edu/dept/EHS/prod/researchlab/bio/docs/Palmer_Bio_protocol.pdf

Category: Mouse Protocols: Mouse Cell Culture Protocols

The procedures involve the isolation and growth of primary cell cultures from rodent and human tissue as well as the use of viral vectors for the introduction and expression of mammalian genes in cells in culture and in live rodents. - [Read Isolation and Growth of Primary Cell Cultures from Mouse Protocol]

Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol - https://catalog.invitrogen.com/index.cfm?fuseaction=iProtocol.unitSectionTree&treeNodeID=9E66368A929753C320BDC7C58DA3FDFD&objectid=66749F0EB1D3C751C86C30427F3C7AB0

Category: Immunology: Mononuclear Cell Isolation Protocols: B Lymphocyte Isolation Protocols

A flow cytometry technique is presented, which results in the selection and isolation of two populations of cells from a complex mixture based on physical properties (e.g., size and internal granularity) and correlated expression of several surface molecules - [Read Isolation of Ly-1+/CD5+ B Cells by Cell Sorting Protocol]

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]

KickStartâ„¢ Protein Expression Kit PDF - http://www.dualsystems.com/pdf/KickStart/P01006_KickStart.pdf

Category: Protein Protocols: Protein Expression Protocols

KickStartâ„¢ Protein Expression Kit PDF. Dual Systems Biotech - [Read KickStartâ„¢ Protein Expression Kit PDF]

Live-Cell Imaging of GFP in Plants - http://www.cshprotocols.org/cgi/content/full/2007/3/pdb.ip31

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]

Measurement of GFP Expression and DNA Content in Permeabilized Cells - http://cyto.mednet.ucla.edu/Protocols/DNA%20and%20GFP%20PI.htm

Category: Reporter Gene Assays: GFP Assay Protocols

Protocol measuring the amount of GFP expression and DNA content in permeabilized cells. Includes: Fix cells with formaldehyde; Permeabilize cells with ethanol; Preparation of Buffered 2% Formaldehyde Solution. - [Read Measurement of GFP Expression and DNA Content in Permeabilized Cells]

Measurement of GFP Expression and DNA Content in Permeabilized Cells Protocol - http://cyto.mednet.ucla.edu/Protocols/DNA%20and%20GFP%20PI.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Data Analysis Protocols

Protocol for the measurement of GFP expression and DNA content in permeabilized cells. Includes: Fix cells with formaldehyde; Permeabilize cells with ethanol; Preparation of Buffered 2% Formaldehyde Solution. - [Read Measurement of GFP Expression and DNA Content in Permeabilized Cells Protocol]

Measurement of Green Fluorescent Protein Expression and DNA Content in Unfixed Cells Protocol - http://cyto.mednet.ucla.edu/Protocols/GFPandDNAHoechst.htm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Flow Cytometry Data Analysis Protocols

Protocol for measurement of green fluorescent protein expression and DNA content in unfixed cells. - [Read Measurement of Green Fluorescent Protein Expression and DNA Content in Unfixed Cells Protocol]

Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte - http://www.biologicalprocedures.com/bpo/arts/1/89/m89.pdf

Category: Nucleic Acid Modification Protocols: DNA Methylation Protocols

Methods for analyzing the role of DNA methylation and chromatin structure in regulating T lymphocyte gene expression. - [Read Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte]

Articles (1-5 of 5)

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Paraffin Embedding Protocol

Paraffin Embedding Protocol for molecular profiling. This Paraffin Embedding Protocol describes the processing of the tissues into sections following ethanol fixation. Molecular profiling (MP) is a technique that is used to visualize the global patterns of RNA expression or protein expression in various cell types and disease processes.

Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

A Phage display expression protocol for antibody expression in 96-well microtiter plates.

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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