purified Protocols

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]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol for 3/7 assay. Includes: Detection of Caspase-3 and -7 Activities in Cell-Based Assays; Detection of Caspase-3 or -7 Activity Using Purified Caspases. - [Read 3-7 Assay Protocol]

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]

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: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol for caspase 8 assay. Includes: Detection of Caspase-8 Activity Using Purified Caspases; Detection of Caspase-8 Activity in Cell-Based Assays. - [Read Caspase 8 Assay Protocol]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Protocol for caspase 9 assay. Includes: Detection of Caspase-9 Activity Using Purified Caspases; Detection of Caspase-9 Activity in Cell-Based Assays. - [Read Caspase 9 Assay Protocol]

Category: PCR Protocols: Real-Time PCR Protocols

Protocol describes the real-time PCR using RNA purified from laser-captured tissues Laser Capture Microdissection (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cells. - [Read cDNA Synthesis and Real-Time PCR Using RNA from Laser-Captured Cells Protocol]

Category: Drosophila Protocols: Drosophila Genetics Protocols

Protocol described here produces chromatin with regularly spaced nucleosomes having physiological nucleosome repeat lengths; something that can be difficult to achieve with purified components. In addition, chromatin assembled with the S-190 Chromatin Assembly Extract contains the ATP-dependent chromatin remodeling factors necessary for efficient transcription. - [Read Chromatin Assembly on Template DNA with Transcription Factors and Drosophila S-190 Chromatin Assembl]

Category: PCR Protocols: PCR Cloning Protocols

Pairs of oligonucleotide primers used in PCR are often designed with restriction sites in their 5' regions.  In many cases, the sites are different in the two primers.  In this case, amplification generates a target fragment whose termini now carry new restriction sites that can be used for directional cloning into plasmid vectors.  The purified fragment and the vector are digested with the appropriate restriction enzymes, ligated together, and transformed into E. coli. - [Read Cloning PCR Products by Addition of Restriction Sites to the Termini of Amplified DNA Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

For microinjection into cells, high-quality plasmid DNA is purified using standard procedures. The resulting preparation is then delivered into the microinjection needle by either a back-filling or a forward-filling approach. - [Read DNA Preparation and Needle Loading for Gene Delivery by Microinjection Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for dot and slot hybridization of purified RNA. Dot blotting of RNA is best carried out using purified preparations of RNA that are denatured with glyoxal or formaldehyde immediately before loading onto a nylon membrane through a vacuum manifold. - [Read Dot and Slot Hybridization of Purified RNA Protocol]

Category: DNA Microarray Protocols

This protocol describes the steps required to produce a cDNA microarray. Gene-specific DNA is produced by PCR amplification of purified template plasmid DNAs from cloned ESTs. The PCR product is purified by ethanol precipitation, thoroughly resuspended in - [Read Fabrication Protocol for DNA Microarrays]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols

A number of density gradient strategies have been developed for the fractionation of human erythrocytes according to their age. As the cells age, so their density tends to increase; reticulocytes therefore tend to have the lowest densities. Reticulocytes have frequently been partially purified on discontinuous gradients of arabinogalactan; the actual density range being quite varied, from quite broad ones. - [Read Fractionation of Human Erythrocytes (Normal or Sickle) and Reticulocytes in Discontinuous Iodixanol]

Category: Pharmacology and Toxicology Protocols: Enzyme Assays Protocols

Homogeneous caspase-3/7 assay. Includes: Detection of Caspase-3/7 Activity in Cell Culture;  Detection of Caspase-3 or -7 Activity in Purified Caspase Preparations; Positive and Negative Cell Culture Controls. - [Read Homogeneous Caspase 3-7 Assay Protocol]

Category: Western Blot Protocols

Protein immunoprecipitation can be a useful preparative step for immunoblotting.  For very rare proteins, the protein of interest can be purified and concentrated by standard immunoprecipitation techniques before immunoblotting.  In addition, protein-protein interactions can be tested with an immunoprecipitating antibody that is specific for one protein of a complex and an immunoblotting antibody that is specific for a second member of the complex. - [Read Immunoblotting: Preparing Immunoprecipitated Proteins Protocol]

Category: Tetrahymena Protozoa Protocols

Protocol describes how to allow the isolation of nuclei from all stages of the Tetrahymena life cycle in high yield with a high degree of purity. This method gives highly purified populations of both micronuclei and macronuclei. - [Read Isolation and Purification of Tetrahymena Nuclei Protocol]

Category: BACs Bacterial Artificial Chromosome Protocols

The procedure for isolation of BAC DNA is scaled-up to accommodate 500-ml cultures, which, on average, yield 20-25 µg of purified BAC DNA. - [Read Isolation of BAC DNA from Large-scale Cultures Protocol]

Category: Antibody Protocols: Antibody Labeling Protocols

Direct labeling of purified antibodies is the method of choice when simultaneously visualizing two or more antibodies of the same species, class, or subclass. This allows the localization of multiple antigens to be compared in the same cell, tissue, or sample. Labeled primary antibodies are also useful for improving background-to-readout ratios, and they can be essential for immunoassays in which good quantification is needed. - [Read Labeling Antibodies with Fluorochromes Protocol]

Category: Cloning Protocols

Protocol for ligating plasmid and target DNAs in low-melting-temperature agarose. Ligation in low-melting-temperature agarose is much less efficient than ligation with purified DNA in free solution and requires a large amount of DNA ligase. The method is used chiefly for rapid subcloning of segments of DNA in dephosphorylated vectors and assembling recombinant constructs. - [Read Ligating Plasmid and Target DNAs in Low-melting-temperature Agarose Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Techniques: Cell Line Preservation Freezing

Following propagation to 1 X 108 cells, lymphoblastoid cells are conveniently stored at -80 degrees C to preserve the high molecular weight DNA in the cells until the DNA is purified. This procedure describes the steps required to harvest and freeze the c - [Read Method: Preparation of Lymphocyte Cell Pellet for Storage]

Category: Microarray Protocols: Microarray Analysis Protocols

Description of gene array analysis and normalization - [Read Microarray Analysis of Highly Purified Human Decidual NK Cells Protocol]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of biotin-labeled target samples and hybridization of these samples to an Affymetrix in situ synthesized oligonucleotide GeneChip array.  The procedure requires a minimum of 5 µg of purified total RNA as starting material. - [Read Microarray Protocol for Affymetrix In Situ Synthesized Oligo Arrays]

Category: Microarray Protocols: Amino-allyl Microarray Methods

Protocol details the preparation of fluorescently labeled target samples (aminoallyl method) and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized]

Category: Microarray Protocols: Microarray DNA Labeling Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides. The procedure requires a minimum of 3 µg of purified total RNA as starting material. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Array]

Category: Microarray Protocols: Microarray Hybridization Protocols

Protocol details the preparation of fluorescently labeled target samples and hybridization of these samples to a microarray of Agilent inkjet-deposited presynthesized oligonucleotides.  The procedure requires a minimum of 3 µg of purified total RNA as starting material. Includes: cDNA Synthesis; Fluorescent cRNA Synthesis; cRNA Precipitation and Cleanup; cRNA Quantification; Hybridization; Washing. - [Read Microarray Protocol for Agilent Inkjet-Deposited Presynthesized Oligo Arrays]