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Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4527

Category: Drosophila Protocols

Protocol for antibody addition to Drosophila specimens and detection using fluorochrome-linked reagents. Fluorochrome-linked reagents should be used when high resolution is needed or if two antigens need to be localized simultaneously. Because of the thickness of fly specimens, detection requires access to a confocal microscope. - [Read Antibody Addition to Drosophila Specimens and Detection Using Fluorochrome-Linked Reagents Protocol]

Contrast in Optical Microscopy - http://microscopy.fsu.edu/primer/techniques/contrast.html

Category: Microscopy Protocols: Optical Microscopy Protocols

When imaging specimens in the optical microscope, differences in intensity and/or color create image contrast, which allows individual features and details of the specimen to become visible. Contrast is defined as the difference in light intensity between the image and the adjacent background relative to the overall background intensity. In general, a minimum contrast value of 0.02 (2 percent) is needed by the human eye to distinguish differences between the image and its background. - [Read Contrast in Optical Microscopy]

Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4350

Category: Cell Culture Protocols: Cell Storing and Cell Freezing Protocols

Protocol provides methods for cryofreezing and subsequent thawing of mammalian cells. Pre-confluent cells are trypsinized, pelleted, resuspended in freezing medium, and gradually frozen. When needed, frozen cells are thawed quickly under running tap water and transferred to growth medium. - [Read Cryopreservation of Mammalian Culture Cells: Preparation and Recovery of Samples Protocol]

Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4673

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Labeling Antibodies with Fluorochromes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/18/pdb.prot4285

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]

Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3284

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Protocol used chiefly to generate large stocks of double-stranded DNA of strains of M13 that are routinely used as cloning vectors. Large amounts of single-stranded DNA of an individual recombinant may occasionally be needed for specific purposes, e.g., to generate many preparations of a particular radiolabeled probe or to construct large numbers of site-directed mutants. - [Read Large-scale Preparation of Single-stranded and Double-stranded Bacteriophage M13 DNA Protocol]

Liquid Phage Lysates Protocol - http://humgen.wustl.edu/hdk_lab_manual/lambda/lambda2.html

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

5 ml liquid lysates are prepared when a small amount of DNA from a large number of lambda clones is needed. The lysates can be made using 10- 20 ul of a stock lysate or a 100-fold amplified phage "macroplaque" as the inoculum. - [Read Liquid Phage Lysates Protocol]

List of Protein Assays - http://www.ruf.rice.edu/~bioslabs/methods/protein/protein.html

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Find a list of assays for the determination of protein concentration in a solution. This list includes the sensitivity range, volume/amount of sample needed, subjective comments on accuracy and convenience, and major interfering agents. Procedural details, equipment requirements, and references are outlined in the individual assay documents. - [Read List of Protein Assays]

Metabolic Labeling & Immunoprecipitation Protocols - http://www.cbrinstitute.org/labs/springer/protocols/jun_35Slabeling_IP.html

Category: Immunology: Immunoprecipitation Protocols: Immunoprecipitation of Radiolabeled Cells Protocols

Cells incorporate 35S-methionine or cysteine during the protein synthesis. Thus it is essential to use Met,Cys-free medium and dialyzed FCS during the labeling. Short period of incubation with 35S-methionine or cysteine will result in radiolabeling (pulse), and additional incubation with excess concentration of unlabeled Met+Cys (chase) is needed for complex glycoproteins like integrins to get expressed as a maturated form. - [Read Metabolic Labeling & Immunoprecipitation Protocols]

Plasmid Transformation of Yeast Colonies Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4146

Category: Yeast Protocols: Yeast Transformation Protocols

Protocol for plasmid transformation of Yeast colonies. Protocol can be used if high efficiency is not needed. - [Read Plasmid Transformation of Yeast Colonies Protocol]

Purification of Antibodies on an Antigen Column Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4284

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Immunoaffinity purification of antibodies is used to purify antigen-specific antibodies from a preparation of polyclonal antibodies. Such purification is commonly needed in the production of antipeptide antibodies, where it is used to concentrate the desired antibodies and separate them from those raised against carrier proteins. It is also used for the more general purpose of removing unwanted, nonspecific binding activity from polyclonal antibody preparations. - [Read Purification of Antibodies on an Antigen Column Protocol]

Articles (1-10 of 12)

Single Stranded Plasmid DNA Isolation Protocol

A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.

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.

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

Probe Preparation for 22 x 40 mm DNA Microarrays

Probe Preparation for 22 x 40 mm DNA Microarrays Protocol.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

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.

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.

Antibodies Phage Expression Protocol Using 96-well Microtiter Plates

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

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.

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.

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