type Protocols

Category: Chromatography Protocols: Immunoaffinity Chromatography Protocols

Antibody Purification (Antiserum or Ascites by Protein A/G Chromatography). Species and Type of Antibody Agarose Rabbit IgG Protein A or Protein G Mouse IgG1 Protein G Mouse IgG2 Protein A or Protein G Mouse IgG3 Protein G Sheep IgG Protein G but binds weakly Rat IgG Protein G but binds weakly Guinea Pig IgG Protein A Dog IgG Protein A Goat IgG Protein G Pig IgG Protein A Hamster IgG Protein G. By Millipore. - [Read Affinity Antibody Purification of Protein A/G Chromatography]

Category: Mouse Protocols: Assembling Chimeras Mouse Protocols

Protocol describes a method for assembling aggregates between diploid embryos. If embryos from a heterozygous mutant intercross are aggregated with wild-type embryos, the resulting chimeras can be used for analyzing mutant phenotypes. - [Read Assembling Aggregates between Diploid Embryos Protocol]

Category: Fungi Protocols: Fungi Genetics Protocols

PCR protocol which uses M. grisea conidia directly for PCR analysis without extraction of DNA. - [Read Assessment of Magnaporthe grisea Mating Type by Spore PCR]

Category: Microinjection Protocols: Microinjection Equipment

This protocol describes an easy method for calibrating micropipette tips that have been pulled in the laboratory. It is essential to estimate the internal diameter of the pulled micropipette tip when adjusting parameters for a new puller or new type of glass tubing. A tip diameter of ~0.3 µm is optimal for the microinjection of mammalian cells in culture (e.g., CHO, PtK1, and COS-7). A 10% increase in diameter increases the delivery rate by more than 30% and can cause cell damage. - [Read Calibration of Micropipette Tips Protocols]

Category: Immunology: Chemotaxis

Chemotaxis Assay, Springer Lab. A chemotaxis assay's function is to assess whether a factor or molecule of interest has chemotactic activity on a motile cell type. Chemotaxis is the ability of a factor to cause the migration of a cell. The chemotactic assay is based on the creation of a chemical gradient of the chemotactic agent which will cause cells to migrate through the gradient towards the chemotactic agent. - [Read Chemotaxis Assay]

Category: Cell Biology and Cell Culture

This chemotaxis assay protocol is based on the premise of creating a gradient of the chemotactic agent and allowing cells to migrate through a membrane towards the chemotactic agent. A chemotaxis assay can determine whether your protein or small molecule of interest has chemotactic activity on a specific cell type. Chemotaxis is then the ability of a protein to direct the migration of a specific cell. - [Read Chemotaxis Assay Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]

Category: Genetics and Genomics: Cancer Genetics Protocols: Single Strand Conformational Polymorphism(SSCP) Protocols

Single-strand conformational polymorphism (SSCP), one of several methods used to scan segments of DNA for mutations, exploits the electrophoretic differences in mobilities between single-stranded mutant and wild-type DNAs. - [Read Detection of Mutations by Single-strand Conformational Polymorphism Protocol]

Category: PCR Protocols

Method uses PCR to amplify and display many cDNAs derived from the mRNAs of a given cell or tissue type.  The method relies on two different types of synthetic oligonucleotides: anchored antisense primers and arbitrary sense primers.  A typical anchored primer is complementary to approx. 13 nucleotides of the poly(A) tail of mRNA and the adjacent two nucleotides of the transcribed sequence. - [Read Differential Display-PCR Protocol]

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Protocol for double immunofluorescence staining for BCL-6. Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved. Includes: unfixed or acetone-fixed specimens; dewaxed, antigen retrieved slides. - [Read Double Immunofluorescence Staining for BCL-6 Protocol]

Category: Recombinant Protein Protocols

Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography. Biological Procedures Online - [Read Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affin]

Category: Histology Protocols: Fixation of Cells Tissues Protocol

There are many variations based on the type of tissue to be examined, whether one is dealing with cell suspensions, biopsies, perfused tissues, or monolayer . Fixation Protocol. OSU Campus Microscopy & Imaging Facility :: The Ohio State University College of Med - [Read Fixation Protocol]

Category: Lipid Protocols

New screening efforts and chemical modifications of existing compounds have been attempted to identify more selective and potent inhibitors. To determine the selectivity of the inhibitors identified during screening efforts we developed gel-elongation assay using crude bacterial lysate directly to determine the target specificities of fatty acid synthesis inhibitors. - [Read Gel-elongation Assay for Type II Fatty Acid Synthesis Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for constant-flow microinjection using the Pneumatic PicoPump (World Precision Instruments). This type of system is very simple and can be assembled on a relatively low budget. In this method, a constant flow of sample is delivered from the tip of the pipette, and the amount of sample injected into the cell is determined by how long the pipette remains in the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Controlled-Flow System Protocol]

Category: Microinjection Protocols: Microinjection of DNA Protocols

This protocol describes a method for pulsed-flow microinjection using the Eppendorf FemtoJet injector and Eppendorf InjectMan; this is the most common type of pulsed-flow microinjection system currently being used. The advantage of this type of system over a controlled-flow system is that much more control is available over the injection parameters, reducing variability in injections. In addition, the system allows a diagonal insertion of the needle into the cell. - [Read Gene Delivery by Direct Injection (Microinjection) Using a Pulsed-Flow System Protocol]

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]

Category: Molecular Model Organisms: Mouse Animal Protocols: Mouse Genotyping Protocols

Genotyping Protocol: MMRRC 425. Assay Type: PCR - can distinguish heterozygous animals from homozygous animals. DNA Extraction: DNA from tail snips was ... - [Read Genotyping Protocol: T1320 mouse]

Category: Primary Cell Isolation and Culture Protocols: Mammalian Cell Culture Protocols

This protocol describes a method for recombining and culturing germ layer fragments. It is useful for testing the inductive properties of fragments from wild-type and mutant mouse embryos. - [Read Germ Layer Explant Recombination Culture Protocol]

Category: Mouse Protocols: Mouse Cell Culture Protocols

Protocol describes a method for recombining and culturing germ layer fragments. It is useful for testing the inductive properties of fragments from wild-type and mutant mouse embryos. - [Read Germ Layer Explant Recombination Culture Protocol]

Category: Signalling Signal Transduction Protocols

This protocol describes assaying kinase activity of a putative kinase using Histone H1 as the substrate. Histone H1 is the canonical kinase substrate in this type of assay. Phosphorylation of Histone H1 is assessed by SDS-Polyacrylamide gel electrophoresis followed by autoradiography. Includes information: H1 Kinase Assay on Individual Xenopus Oocytes; H1 Kinase Assay on Xenopus Egg Extract Samples; H1 Kinase Assay on Tissue Culture Cells; Helpful protocol hints. - [Read Histone H1 Kinase Activity Assay]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to choose wild type laboratory strains of N. crassa for reference and for special uses. - [Read How to Choose Wild Type Laboratory Strains of N. crassa for Reference and for Special Uses]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to convert wild-type spreading growth to colonial. - [Read How to Convert Wild-Type Spreading Growth to Colonial]

Category: Fungi Protocols: Neurospora Protocols

Information and tips on how to determine mating type. - [Read How to Determine Mating Type]

Category: Fungi Protocols: Neurospora Protocols

Information on how to use fluffy testers for determining mating type and for other applications. - [Read How to Use Fluffy Testers for Determining Mating Type and for Other Applications]

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]