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A Method for Assaying Deubiquitinating Enzymes - http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=140113

Category: Protein Protocols: Protein Ubiquitination Protocols

A Method for Assaying Deubiquitinating Enzymes. Lee et al., Biol Proced Online. May 1998. A general method for the assay of deubiquitinating enzymes was described in detail using 125I-labeled ubiquitin-fused Î±NH-MHISPPEPESEEEEEHYC (referred to as Ub-PESTc) as a substrate. - [Read A Method for Assaying Deubiquitinating Enzymes]

A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4117

Category: Genetics and Genomics: Cancer Genetics Protocols: Loss of Heterozygosity Protocols

DNA for analysis is purified using salt precipitation. The method is gentle, limits the breakage of the long chromosomal strands, and avoids the use of phenol and chloroform. It is suitable for use with cultured cells, breast tumor tissue that has been subjected to hormone receptor analysis, and blood samples. The loss of heterozygosity assay is performed using a multiplex PCR, in which one of each primer pair is labeled with a different fluorophor. - [Read A Multiplex PCR Method to Define a Narrow Deleted Chromosomal Region of a Tumor Genome]

Amino-allyl Labeling - http://pga.tigr.org/sop/M004_1a.pdf

Category: DNA Microarray Protocols

This protocol describes the labeling of eukaryotic RNA with aminoallyl labeled nucleotides via first strand cDNA synthesis followed by a coupling of the aminoallyl groups to either Cyanine 3 or 5 (Cy 3/Cy5) fluorescent molecules. Hasseman. TIGR Microarr - [Read Amino-allyl Labeling]

Basic Information on In Vitro Transcription - http://www.ambion.com/techlib/basics/transcription/index.html

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

The ability to synthesize RNA in the lab is critical to many techniques.Radiolabeled and nonisotopically labeled RNA probes, generated in small scale transcription reactions can be used in blot hybridizations and nuclease protection assays. This article includes information on: Requirements For Transcription, RNA Phage Polymerases, Template Options: Plasmids, PCR Products, Oligonuclotides and cDNA, Sense or Antisense, Conventional Or Large Scale Synthesis, Products for In Vitro Transcription. - [Read Basic Information on In Vitro Transcription]

Binding Antibodies to Tissue Sections Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4331

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

CaspaTag Caspase Activity Protocol - http://www.niehs.nih.gov/research/atniehs/core/fc/protocols/caspatag.cfm

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

This assay is based on carboxyfluorescein labeled fluoromethyl ketone (FMK)-peptide inhibitors of caspases. Includes: Working Dilution of FMK-peptide inhibitors; 1X Working Dilution Wash Buffer; Protocol for Flow Cytometry. - [Read CaspaTag Caspase Activity Protocol]

CGH of Biotin Labeled DNA vs Digoxigenin Labeled DNA Protocol - http://waldman.ucsf.edu/Protocols/indirectcgh.html

Category: Cytogenetics Protocols

Protocol for CGH of biotin labeled DNA vs digoxigenin labeled DNA. Includes: Reprecipitate DNA's; Denature slides; Hybridization; Washing and staining. - [Read CGH of Biotin Labeled DNA vs Digoxigenin Labeled DNA Protocol]

CGH of Direct Labeled Test DNA vs Normal DNA Protocol - http://waldman.ucsf.edu/Protocols/directcgh.html

Category: Cytogenetics Protocols

This CGH Protocol is used for DNA of good quality when available in sufficient amounts. We usually do replicate hybridizations using samples labeled "inversely" (reversing the label for test and normal DNA's). If appreciable artifact occurs, then alternative labels are tried. - [Read CGH of Direct Labeled Test DNA vs Normal DNA Protocol]

Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_63-65.pdf

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

This protocol describes how to use DIG Chem-Link to directly label any DNA [e.g. plasmids, PCR products, cDNA prepared from mRNA] or RNA (e.g. total RNA, poly(A)+ mMRNA). The DIG Chem-Link or Biotin Chem-Link may also be used to label oligonucleotides. Includes: Required Purity of DIG Chem-Link Templates; Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Key Product Required for Direct Labeling of DNA or RNA; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_63-65.pdf

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol for Chem-Link labeling of DNA or RNA with DIG or Biotin. Includes: Direct DIG Labeling of mRNA or cDNA with DIG Chem-Link; Estimating the Yield of DIG-labeled Nucleic Acids. - [Read Chem-Link Labeling of DNA or RNA with DIG or Biotin Protocol]

Denaturing Protein Immunoprecipitation from Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4619

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a denaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. The use of denaturing IPs reduces background considerably. - [Read Denaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Detection FISH Protocol - http://www.riedlab.nci.nih.gov/publications/FISH%20Detection.pdf

Category: Cytogenetics Protocols: Fluorescent In Situ Hybridization FISH Protocol

Protocol for Detection (FISH). TRITC, or Avidin Cy-5. For the probes labeled with digoxigenin, we usually first incubate with mouse-anti-digoxigenin, followed by incubation with sheep anti-mouse Cy5.5, or other fluorochrome conjugated antibodies. - [Read Detection FISH Protocol]

Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_216-219.pdf

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol for detection of even-skipped transcripts in Drosophila embryos with PCR/DIG-labeled DNA probes. - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes]

Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_216-219.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of even-skipped transcripts in drosophila embryos with PCR/DIG-labeled DNA probes. This protocol has been used to detect the transcript distribution of a number of genes by in situ hybridization, including evenskipped and seven-up, in whole mount Drosophila embryos, and engrailed Antennapedia in whole mount grasshopper embryos. Includes: Probe labeling; Evaluation of labeling reaction; Preparation of embryos, hybridization and detection. - [Read Detection of Even-Skipped Transcripts in Drosophila Embryos with PCR/DIG-Labeled DNA Probes Protocol]

Detection of HPV 11 DNA in Paraffin-Embedded Laryngeal Tissue with a DIG-Labeled DNA Probe Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_143-148.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of HPV 11 DNA in paraffin-embedded laryngeal tissue with a DIG-labeled DNA probe. Includes: Probe preparation; Pretreatment of slides; Preparation of tissue sections; Hybridization; Washes; Detection of hybrids. - [Read Detection of HPV 11 DNA in Paraffin-Embedded Laryngeal Tissue with a DIG-Labeled DNA Probe Protocol]

Detection of mRNA in Tissue Sections Using DIG-labeled RNA and Oligonucleotide Probes Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_149-163.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA in tissue sections using DIG-labeled RNA and oligonucleotide probes. - [Read Detection of mRNA in Tissue Sections Using DIG-labeled RNA and Oligonucleotide Probes Protocol]

Detection of mRNA on Paraffin Embedded Material of the Central Nervous System with DIG-Labeled RNA - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_164-171.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNA on paraffin embedded material of the central nervous system with DIG-labeled RNA probes. - [Read Detection of mRNA on Paraffin Embedded Material of the Central Nervous System with DIG-Labeled RNA]

Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_189-196.pdf

Category: Cytogenetics Protocols: In Situ Hybridization Tissue Sections Protocols

Protocol for detection of mRNAs on cryosections of the cardiovascular system using DIG-labeled RNA probes. Protocol was optimized from a protocol using 35S-labeled RNA probes. It allows to detect the expression of low abundant mRNAs in the cardiovascular system, e.g. of the proinflammatory cytokine GM-CSF in normal human coronary arteries, and of IL6 and gp130 in human failing hearts. The protocol can be combined with immunohistochemistry. - [Read Detection of mRNAs on Cryosections of the Cardiovascular System Using DIG-Labeled RNA Probes]

Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3791

Category: DNA Protocols: DNA Sequencing Protocols: Dideoxy Mediated Sequencing Protocols

In this protocol asymmetric PCR is used to generate single-strandedDNA templates for dideoxy-sequencing - [Read Dideoxy-mediated Sequencing Reactions Using PCR and End-labeled Primers Protocol]

DIG Labeling of cDNA Probes Protocol - http://home.cc.umanitoba.ca/~frist/Protocols/arrays/DIG_probes.html

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for DIG labeling of cDNA probes. Includes: Incorporation DIG during PCR; Estimating the yield of DIG labeled probes; DNA Dot Blotting; Prehybridization and Hybridization. - [Read DIG Labeling of cDNA Probes Protocol]

DIG Labeling of cDNA Probes Protocol - http://home.cc.umanitoba.ca/~frist/Protocols/arrays/DIG_probes.html

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Direct Selection of cDNAs with Large Genomic DNA Clones Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4077

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

The goal of this method is to identify transcriptionally active genes in cloned segments of genomic DNA. The protocol uses hybridization and affinity purification to recover biotin-labeled cDNAs that bind to a 500-kb segment of human DNA cloned in a BAC vector. However, the method can be easily adapted to other clones of genomic DNAs cloned in high-capacity vectors. - [Read Direct Selection of cDNAs with Large Genomic DNA Clones Protocol]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol describes here a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. Includes: In situ hybridization with DIG-labeled probes; Detection of DIG-labeled probes; Fluorescence microscopy. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System]

DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol - http://www.roche-applied-science.com/PROD_INF/MANUALS/InSitu/pdf/ISH_124-126.pdf

Category: Nucleic Acid Modification Protocols: DIG Biotin Labeled Probes Protocols

Protocol describes a high sensitivity indirect detection procedure for DIG-labeled hybridization probes. The procedure uses the components of the HNPP Fluorescent Detection Set to form a fluorescent precipitate of HNPP (2-hydroxy-3-naphthoic acid-2’-phenylanilide phosphate) and Fast Red TR at the site of hybridization. This procedure can be used to detect single copy sequences as small as 1 kb on human metaphase chromosomes. - [Read DNA In Situ Hybridization with an Alkaline Phosphatase-Based Fluorescent Detection System Protocol]

DNA Sequencing Methods - http://www.genome.ou.edu/protocol_book/protocol_partIV.html#IV.C

Category: DNA Protocols: DNA Sequencing Protocols

Methods for DNA sequencing. Includes: Bst-catalyzed radiolabeled DNA sequencing; Radiolabeled sequencing gel preparation, loading, and electrophoresis; Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye primers; Taq-polymerase catalyzed cycle sequencing using fluorescent-labeled dye terminator reactions; Sequenase[TM] catalyzed sequencing with dye-labeled terminators; etc. - [Read DNA Sequencing Methods]

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.

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.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.