conditions Protocols

Category: Xenopus Protocols: Xenopus Egg Extracts Protocols

Protocol for HSS depletion conditions for XKCM1. - [Read HSS Depletion Conditions for XKCM1 Protocol]

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

Coimmunoprecipitation is most commonly used to test whether two proteins of interest are associated in vivo, but it can also be used to identify novel interacting partners of a target protein. In both cases, the cells, which may have been labeled with [35S]methionine, are harvested and lysed under conditions that preserve protein-protein interactions. The target protein is specifically immunoprecipitated from the cell extracts, and the immunoprecipitates are fractionated by SDS-PAGE. - [Read Identification of Associated Proteins by Coimmunoprecipitation Protocol]

Category: Cytogenetics Protocols: DIG Biotin Labeled Probe Protocols

Protocol for the identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides; Detection of DIG-labeled oligonucleotides with fluorescently labeled anti-DIG Fab fragments; Detection of DIG-labeled oligonucleotide.
 - [Read Identification of Single Bacterial Cells using DIG-Labeled Oligonucleotides Protocol]

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

Protocol for identification of single bacterial cells using DIG-labeled oligonucleotides. Includes: Organisms and growth conditions; Cell fixation and preparation of cell smears; DIG labeling of oligonucleotides with DIG-ddUTP; In situ hybridization using digoxigenin-labeled oligonucleotides. - [Read Identification of Single Bacterial Cells Using DIG-Labeled Oligonucleotides Protocol]

Category: Cell Culture Protocols: Cell Immortalization Protocols

Cultivating animal cells in the laboratory is an indispensable technique for cell biologists. However, most normal primary cell lines, while faithfully reproducing the phenotype of their tissue of origin, do not grow indefinitely in culture. After a series of population doublings (the number of which varies by species, cell type, and culture conditions) primary cells enter a state where they no longer divide. - [Read Immortalization of Cells in Culture]

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols

After fixation, frozen sections are immunostained under RNase-free conditions using a rapid three-step streptavidin-biotin technique followed by dehydration. The immunostained sections are ready for LCM. Includes: Development of Immuno-LCM. - [Read Immuno-Laser Capture Microdissection Protocol]

Category: Immunohistochemistry Protocols

Describes two methods for using the immunoperoxidase reaction to localize antigens at the electron microscope level; one for adherent cultured cells and one for tissue sections. The reaction conditions are first optimized at the light microscope level and then adapted for EM level observation. These methods allow for reliable detection of antigens at the cell surface, within the cell, and especially in membrane bounded organelles. - [Read Immunoperoxidase Methods for Localization of Antigens in Cultured Cells and Tissues]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation with anti-phosphotyrosine:biotin conjugates. Includes: Denaturing Conditions; Native Conditions; Pre-clearing. - [Read Immunoprecipitation with Anti-Phosphotyrosine:Biotin Conjugates Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation with antibody:agarose conjugates. Includes: Denaturing Conditions; Native Conditions; Pre-clearing. - [Read Immunoprecipitation with Antibody:Agarose Conjugates Protocol]

Category: Immunology: Immunoprecipitation Protocols

Protocol for immunoprecipitation with soluble antibodies. Includes: Denaturing Conditions; Native Conditions; Pre-clearing. - [Read Immunoprecipitation With Soluble Antibodies Protocol]

Category: Immunology: Immunoprecipitation Protocols

For many sources of antigens, one useful method of lysis is to treat cells with harsh, denaturing solutions to release most of the protein antigens, as described here. The lysates are then diluted to reduce the denaturing conditions to levels that are suitable for the formation of antibody-antigen complexes. The resulting solution is precleared prior to immunoprecipitation. - [Read Immunoprecipitation: Denaturing Lysis Protocol]

Category: Immunohistochemistry Protocols

Immunostaining conditions. Includes: Antibody, Host, Source, Cat #, Antigen Retrieval, Block, Diluent, Ab Dilution and 2° antibody. - [Read Immunostaining Conditions]

Category: PCR Protocols: In-situ PCR Protocols

Amplification and Detection in a Cellular Context. Methodology, in-situ detection methods, reaction Conditions, introduction. Ernest F. Retzel et al., University of Minnesota. - [Read In-Situ PCR Protocol]

Category: Tetrahymena Protozoa Protocols

Mature Tetrahymena cells of opposite mating types are starved under appropriate salt conditions. The mating types are then combined to costimulate through cell-cell interaction. Loose pairs and then firm, irreversible pairs of cells of opposite mating types form. This method consistently results in a high percentage of pairing (usually greater than 80%) and good synchrony. - [Read Induction of Conjugation in Tetrahymena Protocol]

Category: Cloning Protocols

Protocol can be used to optimize ligation conditions for difficult to clone (e.g. very large) fragments. The principle is to independently characterize the ligation kinetics of the vector and insert DNA fragments and then to combine them in optimal ratios. - [Read Ligation Optimization Protocol]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols: Long PCR Protocols

General Guidelines for Long PCR Conditions and Enzyme Mixtures. Pete Estep. Harvard. - [Read Long PCR Reagents and Guidelines]

Category: PCR Protocols: Quantitative PCR Protocols

General guidelines for long-PCR conditions and enzyme mixtures. Efficient long-PCR results from the use of two polymerases: a non-proofreading polymerase is the main polymerase in the reaction, and a proofreading polymerase (3' to 5' exo) is present at a lower concentration. Includes: For PCR with low-complexity templates (e.g., plasmid and cosmid inserts); For PCR with moderate-complexity templates (e.g., bacterial genomic DNA); For PCR with high-complexity templates (e.g., human genomic DNA). - [Read Long-PCR Reagents and Guidelines]

Category: Tetrahymena Protozoa Protocols

Long-term storage of unfrozen Tetrahymena is convenient when a laboratory is not using the cells frequently; however, cells maintained under these conditions often take a couple of days of incubation in culture medium to recover. - [Read Long-Term Storage of Unfrozen Tetrahymena Cultures in Soybean Medium Protocol]

Category: Protein Protocols: Lowry Protein Assay

Lowry Protein Assay. The Lowry procedure is one of the most venerable and widely-used protein assays, being first described in 1951 [Lowry et al., J. Biol. Chem. 193: 265-275 (1951)]. Under alkaline conditions, copper complexes with protein. When folin phenol reagent (phospho-molybdic-phosphotungstic reagent) is added, the Folin-phenol reagent binds to the protein. Bound reagent is slowly reduced and changes color from yellow to blue. P.J. Hansen, Dept. of Animal Sciences, University of Florida. - [Read Lowry Protein Assay]

Category: Cell Biology and Cell Culture: Cell Adhesion Protocols

Many proteins and molecules promote cell adhesion including several cell surface carbohydrate binding proteins. Cell adhesion measurements on 96-well microtiter plate format are difficult due to the shear forces generated by washing the wells. The protocol here introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity. This eliminates uncontrolled shear forces and passage of adherent cells through a liquid/air interface. John L. Magnani~GlycoTech Corporation. - [Read Measurement of Cell Adhesion Under Static Conditions]

Category: Yeast Cell Culture Protocols: Yeast Media, Solutions and Stocks Protocols

The yeast, Saccharyomyces cerevisiae, has become an important organism in molecular, biochemical, and genetic analysis. The organism has specific requirements for growth under a variety of conditions. The media, both liquid and solid, simple, define, and complex are describe in this unit. Also included are methods for handling, storing, and shipping stock of yeast. - [Read Media and Culture of Yeast Protocol]

Category: Cell Biology and Cell Culture: Cell Culture Growth Media: Mammalian Cell Culture Growth Media Protocols

The culture medium is an essential component of the in vitro environment and must be selected or designed with care. This protocol provides guidelines for design of serum-containing and serum-free media, selective and specialty media, and media for growth under special conditions such as soft-agar growth. - [Read Media for Culture of Mammalian Cells Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Discusses the effects of various components of the hybridization solution on the rate of renaturation and thermal stability of DNA hybrids free in solution. Includes: The main parameters that influence hybridization; Additional hybridization variables; Competition in situ hybridization; Oligonucleotide hybridization; Standard in situ hybridization conditions. - [Read Nucleic Acid Hybridization General Aspects]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of absorption condition for dye-ligand affinity chromotography. Generally, low pH and low ionic strength, absence of phosphate ions, and the presence of divalent metals ions increase the binding of proteins to immobilized triazine dyes. - [Read Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol]

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of elution condition for dye-ligand affinity chromotography. Elution methods used in dye-ligand affinity chromatography may be either selective or nonselective in nature.  Usually, selective elution methods are applied in combination with group-specific adsorbents, such as dye-ligand adsorbents, and nonselective elution methods are used in combination with highly specific adsorbents. - [Read Optimization of Elution Conditions for Dye-Ligand Affinity Chromatography Protocol]