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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]

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]

Gene Delivery to Skin Using Biolistics Protocol - http://www.cshprotocols.org/cgi/content/short/2006/1/pdb.prot4446

Category: Gene Delivery Protocols: Biolistics Protocols

The technique has many advantages—plasmids may be used for delivery, DNA theoretically can be delivered to any cell type, and genes may be delivered to cells in vitro, ex vivo, or in vivo. DNA-coated gold particles are distributed evenly along the length of the tubing, which is subsequently cut into short sections of cartridges to be used in a gene gun. The Helios Gene Gun uses a pulse of helium to launch the DNA-coated particles, spreading them onto the target cells. - [Read Gene Delivery to Skin Using Biolistics Protocol]

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]

Nondenaturing Protein Immunoprecipitation from Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4620

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a nondenaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. However, the nondenaturing protocol is useful when one wishes to separate soluble from insoluble proteins, or when the antibody being used recognizes a native epitope. - [Read Nondenaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Pulse-Chase Assay for Measuring Protein Stability in Yeast Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4641

Category: Yeast Protocols: Yeast Protein Protocols

This assay directly measures the stability of a specific protein in yeast. Following pulse-labeling of yeast cells, the target protein is recovered by denaturing immunoprecipitation. - [Read Pulse-Chase Assay for Measuring Protein Stability in Yeast Protocol]

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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.