intact Protocols

Category: Cell Organelle Protocols: Mitochondria Protocols

The technique of JC-1 staining has been developed with the intent to detect DY in intact, viable cells. For this purpose JC-1 acts as a marker of mitochondrial activity, since the formation of J-aggregates, which give red emission, is reversible. Cells with high DY are those forming J-aggregates, thus showing high red fluorescence. On the other hand, cells with low DY are those in which JC-1 maintains (or re-acquire) monomeric form, thus showing only green fluorescence. - [Read Analysis of Mitochondrial Membrane Potential with the Sensitive Fluorescent Probe JC-1]

Category: Molecular Biology Protocols: Carbohydrate Protocols

Protocol detects specific cell adhesion to glycolipids resolved on TLC plates. Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates. Ronald L. Schnaar~Professor, Johns Hopkins University Medical School, Baltimore, Maryland. Glycotech. - [Read Carbohydrate-Specific Adhesion of Intact Cells to Resolved Glycolipids on TLC Plates]

Category: Plant Biology Protocols

Protocol can be used for clearing intact non-ovule materials of arabidopsis, which can then be observed under Normarski optics. This is an efficient way to analyse root, seedling even flower development without sectioning. This protocol could also be used for clearing GUS stained material, after chlorophyll is removed by 70% ethanol. - [Read Clearing Arabidopsis Non-Ovule Materials With the HCG Solution Protocol]

Category: Plant Biology Protocols

Protocol can be used for clearing intact seeds of arabidopsis, which can then be observed under Normarski optics. This is an efficient way to analyse embryo and endosperm development without sectioning. - [Read Clearing Arabidopsis Ovule Materials with the Hoyer's Solution Protocol]

Category: Yeast Protocols: Yeast Genetics Protocols: YACs Protocols

Protocol describes methods for isolation of DNA from a strain of S. cerevisiae carrying a recombinant YAC. Because the linear YAC DNAs are sensitive to shearing forces, pipettes with wide-bore tips should be used to transfer DNAs. The method is suitable for preparing DNA that will be used for agarose gel electrophoresis, Southern blotting, subcloning, genomic library construction, PCR, or other methods that do not require intact high-molecular-weight DNA. - [Read Growth of S. cerevisiae and Preparation of DNA Protocol]

Category: Yeast Protocols: Yeast Nucleic Acid Protocols: Yeast DNA Isolation Protocols

Protocol that isolates intact, high molecular weight DNA from yeast cells for subcloning and rare cutting restriction enzyme analysis. Using this protocol one can expect a yield of 100-200 µg of DNA per prep. - [Read High Molecular Weight Yeast Liquid DNA Preparation Protocol]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

The same GFP-tagged actin construct used in cell transfection experiments has been used to produce transgenic mice. Transgenic animals allow the imaging of brain tissue in the intact animal, as acutely cut slices or as organotypic slice cultures. They also serve as a source of cells for imaging neurons at high resolution in dispersed low-density cell culture. In contrast to cells transfected in culture, where the level of actin-GFP expression in neurons varies considerably, transgenic mice... - [Read Imaging Actin in Tissue Slices from Transgenic Mouse Brain Protocol]

Category: Cell Biology and Cell Culture: In Vitro Reconstitution Protocols

This protocol describes the use of SEMV cells (a cell line derived from ram seminal vesicles) in studies into prostaglandin H synthase-mediated metabolism of xenobiotics in intact cells. - [Read In Vitro Model For Studies Of Prostagland H Synthase (PHS)- Mediated Genotoxicity Of Xenobiotics]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol for the isolation of infectious HIV-1. Includes: HIV-1 capture strategy; Protocol for the isolation of HIV-1 virions; Magnetic labeling; Magnetic separation; Elution option A for virion lysate; Elution option B for intact virions. - [Read Isolation of Infectious HIV-1 Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: DNA Staining Protocols for Flow Cytometry

This protocol describes a method for quantitative measurement of DNA using propidium iodide (PI) staining and flow cytometry. PI stains all double-stranded regions of both DNA and RNA by intercalating between the stacked bases of the double helix. PI cannot penetrate an intact cell membrane; therefore, cells are fixed prior to staining. The ethanol-fixed cells can be stored unstained at 4°C for days, or even weeks, and then stained and analyzed. - [Read Measurement of DNA Content Using Propidium Iodide (PI) Staining of Fixed Whole Cells Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

Green fluorescent protein is commonly used to monitor gene expression and protein trafficking within intact cells. The Monster Green® Fluorescent Protein is encoded by an improved synthetic version of the green fluorescent protein gene originally cloned from Montastrea cavernosa (Great Star Coral). - [Read Monster Green® Fluorescent Protein Assay]

Category: Cell Biology and Cell Culture

Cell fractionation protocol to yield intact plant protoplasts. Technique used to purify protoplasts from the grass Glyceria fluitans. Protocol includes: Determination of leaf osmolality; Sterilization. - [Read Plant Cell Purification of Intact Plant Protoplasts]

Category: Nucleic Acid Electrophoresis Protocols: Pulsed Field Gel Electrophoresis Protocols

Protocol for preparation of DNA for pulsed-field gel electrophoresis: isolation of intact DNA from yeast. Yeast cells are first treated enzymatically to break down the cell walls and then resuspended in low-melting-temperature agarose plugs.  The DNA is liberated by infusing the plugs with lysis buffer and proteases.  This method is used to prepare both conventional and artificial yeast chromosomes. - [Read Preparation of DNA for Pulsed-field Gel Electrophoresis: Isolation of Intact DNA from Yeast]

Category: Plant Biology Protocols: Plant Proteins, Peptides and Antigens

The activity of ß-glucuronidase (GUS) can be accurately determined in intact plant tissue using 4-methylumbelliferyl ß-D-glucuronide (4-MUG) as a substrate. Upon hydrolysis by GUS, the fluorochrome 4-methyl umbelliferone (4-MU) is produced. This method is based on the permeability of both 4-MUG and 4-MU through plant tissue. It consists of incubation of the tissue with the reagent and quantification of the fluorescence emitted by 4-MU in the solution. GUS activity in each sample can be... - [Read Quantitative GUS Activity Assay in Intact Plant Tissue Protocol]

Category: Cell Biology and Cell Culture

Protocol for the isolation of the lipid-rich microdomains of the plasma membrane, notably caveolae and lipid rafts. Methods for the isolation of lipid rafts are based on the insolubility of these structures in the nonionic detergent TritonX-100. Either the intact cells are treated with a detergent-containing solution or a post-nuclear supernatant is prepared from a cell homogenate and then Triton X-100 is added to this supernatant. - [Read S20 Purification of detergent-insoluble lipid rafts from cells and tissues.]