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(LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4107

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages. - [Read (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel]

3' Rapid Amplification of cDNA Ends (RACE) Protocol - http://www.nottingham.ac.uk/~mbzspd/methods/3RACE_PCR.html

Category: RNA Protocols: 3' RACE Rapid Amplification of cDNA Ends Protocols

PCR cycle steps, protocol using SuperScript II reverse transcriptase (Life Technologies) and Taq polymerase, Gene specific primer, T17 Adapter primer and an Adapter primer. Dr.Dawson, Nottingham. - [Read 3' Rapid Amplification of cDNA Ends (RACE) Protocol]

3'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4130

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "3'-end" partial cDNA clones, mRNA is reverse-transcribed using a "hybrid" primer (Qtotal, QT) that consists of two mixed bases (GATC/GAC followed by [T]17) and a unique 35-base oligonucleotide sequence (QI-QO). Amplification is then performed using a primer containing part of this sequence (Qouter, Qo) (which now binds to each cDNA at its 3'-end) and a primer derived from the gene of interest, GSP1 (gene-specific primer 1). - [Read 3'-End cDNA Amplification Using Classic RACE Protocol]

5'-End cDNA Amplification Using Classic RACE Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/14/pdb.prot4131

Category: PCR Protocols: cDNA Synthesis Protocols

To generate "5'-end" partial cDNA clones using classic RACE, the first-strand products are generated by reverse transcription (primer extension) from a known gene-specific primer (GSP-RT). Then, a poly(A) tail is appended using terminal deoxynucleotidyltransferase (Tdt) and dATP. Amplification is carried out using three primers. - [Read 5'-End cDNA Amplification Using Classic RACE Protocol]

96-Well Confirmation Yeast PCR Protocol - http://sequence-www.stanford.edu/group/yeast_deletion_project/96_well_confirmation.html

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

Protocol for 96-well confirmation Yeast PCR. Includes: Clonal purification; Generate a master plate (96-well format); Making a frozen backup stock; Confirmation PCR for one Row; ORF Specific Confirmation PCR --> "A-B" primers (upstream junction); Transfer template DNA to multiwell PCR plate; Prepare and dispense master mix for A-B PCR. - [Read 96-Well Confirmation Yeast PCR Protocol]

A high yield affinity purification method for specific RNA-binding proteins. - http://www.pubmedcentral.gov/pagerender.fcgi?tool=pmcentrez&pageindex=1&artid=330202

Category: RNA Protocols: RNA-binding Protein Purification

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Neupert 1990. - [Read A high yield affinity purification method for specific RNA-binding proteins.]

A Sensitive ELISA-Based Assay for Glycosyltransferases - http://www.glycotech.com/protocols/Proto4.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

The method allows the detection and quantification of glycosyltransferase activity using an ELISA-based procedure and carbohydrate-specific monoclonal antibodies. Avoids the use of radiolabeled substrates. Bruce A. Macher~Professor of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA - [Read A Sensitive ELISA-Based Assay for Glycosyltransferases]

A specific endpoint assay for ubiquitin. - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=304457

Category: Protein Protocols: Protein Ubiquitination Protocols

A specific endpoint assay for ubiquitin. Rose et al., Proc Natl Acad Sci U S A. 1987. Simple endpoint assays for free ubiquitin (Ub) and for the Ub-activating enzyme are described. . - [Read A specific endpoint assay for ubiquitin.]

An Alternatively Spliced Isoform of t(8;21) Transcript Promotes Leukemogenesis - http://www.natureprotocols.com/2006/08/03/an_alternatively_spliced_isofo.php

Category: Genetics and Genomics: Cancer Genetics Protocols

Describe the methods to identify and quantitate the specific A/E9a transcript in t(8;21) patients samples relative to the AML1-ETO transcript encoding the well known full-length 752 amino acid AML1-ETO protein (AE). Includes: RNA preparation and RT-PCR; Relative quantitation of the AE9a and the AE transcripts. - [Read An Alternatively Spliced Isoform of t(8;21) Transcript Promotes Leukemogenesis]

Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000230.pdf?pid=PP00000230

Category: RNAi Technology Protocols

Protocol describes the preparation of an shRNA-expressing Gateway entry vector. This process is preceded by the design and synthesis of target gene-specific sense and antisense oligonucleotides which, when annealed, will constitute an shRNA cassette. - [Read Annealing, Cloning, and Sequencing of shRNA Linkers into pEN_H1 Plasmids Protocol]

Antibody Addition and Detection for Staining Caenorhabditis elegans Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4523

Category: C. elegans Protocols: C. elegans Staining Protocols

Extreme care should be used to identify and verify positive reactions, however, because cross-reactions are common. Counterstaining is essential for examining worms by immunofluorescence and is used to identify the exact cell in which an antigen appears. Methods for counterstaining include labeling all cells with a fluorescent dye that is specific for nucleic acids (e.g., DAPI or propidium iodide) and using GFP driven by tissue-specific promoters. - [Read Antibody Addition and Detection for Staining Caenorhabditis elegans Protocol]

Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000261.pdf?pid=PP00000261

Category: Transfection Protocols: Stable Transfection Protocols

The AfCS is utilizing antisense technology to manipulate signaling protein expression in the RAW 264.7 macrophage-like cell line. This can be achieved by the transfection of gene-specific antisense oligonucleotides (ASOs). The following procedure involves the transfection of ASOs into RAW 264.7 cells using FuGENE 6 transfection reagent. Subsequently, the isolated total RNA or protein from these transfected cells can be used to assess the level of mRNA or protein knockdown, respectively. - [Read Antisense Oligonucleotide Transfection of RAW 264.7 Cells with FuGENE 6 in a 24-Well Dish]

Applications of SKY in Cancer Cytogenetics - http://www.utoronto.ca/cancyto/protocols_software/cytogenetics/SKY.html

Category: Cytogenetics Protocols: Spectral Karyotyping SKY Protocols

SKY has been applied to various tumor groups including hematological malignancies, sarcomas, carcinomas and brain tumors, with the intent of identifying specific chromosomal abnormalities that may provide insight to the genes involved in the disease process as well as identifying recurrent cytogenetic markers for clinical diagnosis and prognostic assessment. - [Read Applications of SKY in Cancer Cytogenetics]

Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4450

Category: Gene Delivery Protocols: DNA Nanoparticles Protocols

This protocol describes a stepwise procedure to prepare nucleic acids encapsulated in a polyethylene glycol (PEG)-shielded nanolipoparticle (NLP) that contain a bioresponsive lipid and ligand. This process provides several advantages for systemic gene delivery. The in vivo circulation time is extended. Also, low pH-sensitive lipids enhance DNA unpacking and endosomal escape. Finally, ligands inserted into the NLP surface can target gene delivery to specific tissues or cells in vivo. - [Read Bioresponsive Targeted Charge Neutral Lipid Vesicles for Systemic Gene Delivery Protocol]

Blocking of Unwanted Non-Specific Staining Protocol - http://icg.cpmc.columbia.edu/cattoretti/Protocol/immunohistochemistry/NonSpecificBlocking.html

Category: Immunohistochemistry Protocols: Blocking Protocols

Protocol for blocking of unwanted non-specific staining. Includes: Blocking of endogenous enzymes; Blocking of endogenous fluorochromes; Blocking endogenous biotin; Blocking of endogenous Fc blocking; Blocking of crossreactive antigens in the tissue. - [Read Blocking of Unwanted Non-Specific Staining Protocol]

Blocking of unwanted non-specific staining in Immunofluorescence. - http://icg.cpmc.columbia.edu/cattoretti/Protocol/immunohistochemistry/NonSpecificBlocking.html

Category: Molecular Imaging: Immunofluorescence Microscopy

Blocking of unwanted non-specific staining in Immunofluorescence. Blocking of endogenous enzymes, Blocking of endogenous fluorochromes, Blocking endogenous biotin , Blocking of endogenous Fc blocking, Blocking of crossreactive antigens in the tissue. Cattoretti. Columbia University - [Read Blocking of unwanted non-specific staining in Immunofluorescence.]

Blocking Solutions Protocols - http://icg.cpmc.columbia.edu/cattoretti/Protocol/main_page_reagents.html#Blocking%20solutions

Category: Immunohistochemistry Protocols: Blocking Protocols

Protocols for blocking solutions. Includes: Endogenous peroxidase blocking solution (H2O2-Azide); Non-specific antibody background blocking solution: swine serum; Non-specific antibody background blocking solution: MILK; Enzymatic antigen retrieval solution; Unconjugated, biotin- and fluorochrome-conjugated antibody (primary) solutions; Unconjugated, biotin- and fluorochrome-conjugated antibody (secondary) solutions; Enzyme-conjugated secondary antibody solutions.; etc.. - [Read Blocking Solutions Protocols]

Blocking Unwanted Non-Specific Staining Protocol - http://www.ihcworld.com/_protocols/general_IHC/ihc_blocking.htm

Category: Immunohistochemistry Protocols: Blocking Protocols

Protocol for blocking of unwanted non-specific staining. Includes: Blocking of endogenous enzymes; Blocking of endogenous fluorochromes; Blocking endogenous biotin; Blocking of endogenous Fc blocking; Blocking of cross reactive antigens in the tissue. - [Read Blocking Unwanted Non-Specific Staining Protocol]

C. elegans Gene Knockout Protocol - http://www.zoology.ubc.ca/~alorch/koprotocol.htm

Category: C. elegans Protocols: C. elegans Genetics Protocols

The procedure is to mutagenize a large population of worms with trimethylpsoralen and UV irradiation, set up 1152 subpopulations, screen DNA made from this library for deletions in specific genes by nested PCR, and then to recover single worms carrying the deletions through a sib-selection process. - [Read C. elegans Gene Knockout Protocol]

Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates - http://www.glycotech.com/protocols/Proto9.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

Protocol detects specific cell adhesion to glycolipids resolved on TLC plates. Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates. Ronald L. Schnaar~Professor, Johns Hopkins University Medical School, Baltimore, Maryland. Glycotech. - [Read Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates]

Cell Lysate Extracts - http://www.upstate.com/misc/protocol_detail.q.prot.e.CellLysate_Prep.a.name.e.Cell_Lysate_Extracts-General_Protocols

Category: Protein Protocols: Protein Extraction From Cells

Cell Lysate Extracts. Great protocols for cell lysis preparation from a variety of cell types. There are numerous methods of cell stimulation and lysis. For a given protein, Upstateâ€™s Laboratories determine the specific treatment upon initial testing of its products. It is important to select the correct cell line, stimulation procedure (if any), and lysis protocol. Upstate. - [Read Cell Lysate Extracts]

Channel Specific Techniques - http://physiologie.uni-ulm.de/angewandte-physiologie/FuN/pages/principles_behind_the_techniques.htm

Category: Neuroscience Protocols: Patch Clamping Protocols

Principles behind the techniques: channel specific techniques. - [Read Channel Specific Techniques]

Characterization of Cells by Flow Cytometry Protocol - http://www.signaling-gateway.org/data/cgi-bin/ProtocolFile.cgi/afcs_PP00000018.pdf?pid=PP00000018

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol uses specific antibodies coupled to one of four fluorochromes: fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), peridinin chlorophyll-a (PcP), and allophycocyanin (APC). These fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer. - [Read Characterization of Cells by Flow Cytometry Protocol]

Chemotaxis Assay Protocol - http://www.cbrinstitute.org/labs/springer/protocols/melissa_chemotaxis.html

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]

Chromatin IP (CHIP assay) - http://www.fhcrc.org/science/labs/breeden/Methods/chromatinIP.html

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

ChIP assay protocol with 2 steps: in vivo formaldehyde cross-linking of whole cells and protein-protein and protein-DNA interactions, followed by immunoprecipitation of protein-DNA complexes with specific antibodies from sonicated extracts. Breeden Lab - [Read Chromatin IP (CHIP assay)]

Articles (1-7 of 7)

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.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

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.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol

3' Rapid Amplification of cDNA Ends RACE Using PCR Protocol. This protocol contains the steps for 3' end rapid amplification of mRNA by PCR. The first-strand cDNA is synthesized from total or poly(A+) RNA by priming from the poly-A tail of the mRNA using a oligo (dT) adaptor primer. The cDNA is then amplified via PCR using a gene-specific primer and an adaptor primer.

Mast Cell Staining Protocol

A simple and concise mast cell staining protocol for cell histology.

Electrotransformation of BMH 81-17mut S for Isolating Site-Directed dsDNA Mutants

This protocol describes the electroporation of the BMH 81-17 mut S strain that is recommended for tranformation of the site directed mutagenesis of dsDNA (See Protocol on Site-Directed Mutagenesis on Double Stranded DNA). BMH 81-17 mut S are a mismatch repair defective (mut S) Escherichia coli strain. The probability that the two mutations will cosegregate during the first round of DNA replication is increased in this strain.

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