separated Protocols

Category: Protein Protocols: Protein Quantitation Concentration Protocols: Bradford Protein Assay Protocols

Bradford Protein Assay Spectrophotometry.  Includes spectrophotometry information and the Bradford protein assay: A spectrophotometer or colorimeter makes use of the transmission of light through a solution to determine the concentration of a solute within the solution.  A spectrophtometer differs from a colorimeter in the manner in which light is separated into its component wavelengths.  A spectrophotometer uses a prism to separate light and a colorimeter uses filters. - [Read Bradford Protein Assay Spectrophotometry]

Category: Proteomic Protocols: 2-D Gel Electrophoresis

In gel trypsin digestion for Mass spectrometry. (According to EMBL Protein and Peptide Group, 1997) - [Read In-Gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresis]

Category: Cell Biology and Cell Culture

Purification protocols of the viruses: HIV-1, Lassa virus, oncornavirus and other retroviruses. Protocol uses an iodixanol gradient in a sedimentation velocity mode to purifyHIV-1 virions without affecting the infectivity of the virus. In rate-zonal iodixanol gradients the HIV-1 was effectively separated both from Vif and from the microvesicles. - [Read M5 Velocity (rate zonal) gradients for purification and assembly analysis of viruses.]

Category: Nucleic Acid Purification Protocols

Preparations of RNA containing an mRNA of interest are hybridized to a complementary single-stranded DNA probe.  At the end of the reaction nuclease S1 is used to degrade unhybridized regions of the probe, and the surviving DNA-RNA hybrids are then separated by gel electrophoresis and visualized by autoradiography or Southern hybridization.  Method used to quantitate RNAs, to map the positions of introns, and to identify the locations of 5' and 3' ends of mRNAs on cloned DNA templates. - [Read Mapping RNA with Nuclease S1 Protocol]

Category: Reporter Gene Assays: Cat Assays Protocols

In this protocol, extracts prepared from cells transfected with a chloramphenicol acetyltransferase (CAT) reporter plasmid are incubated with radiolabeled chloramphenicol. The acetylated products generated by the action of CAT are separated from the unmodified drug by thin-layer chromatography and quantitated by scraping the spots from the thin-layer plates and counting them by scintillation spectroscopy. - [Read Measurement of CAT in Extracts of Mammalian Cells Using Thin-layer Chromatography]

Category: Yeast Cell Culture Protocols: Yeast Sporulation Protocols

Zygotes can be identified by their unique morphology. They can be easily separated away from nonmated cells using a micromanipulator. This method provides an alternative to the selection of diploid cells on a medium that prevents the growth of haploid parent cells. - [Read Picking Zygotes Protocol]

Category: Cytogenetics Protocols

An ideal method of tissue preparation ensures both good specimen morphology and that the target molecules are in the optimum state for probe access and hybridization. DNA:DNA in situ hybridization is usually carried out on chromosome spread preparations where chromosome and nuclei are released from cells and spread on a glass microscope slide. This method yields well separated and enlarged chromosomes with good morphology which can be analyzed in transmitted light or fluorescence microscopes. - [Read Preparation of Chromosome Spreads]

Category: Nucleic Acid Purification Protocols

Radiolabeled nucleic acids, including oligonucleotides, can be separated from unincorporated radiolabel by quantitative, differential precipitation with the cationic detergent cetylpyridinium bromide (CPB).  The nucleic acids are first precipitated from aqueous solution with CPB. - [Read Purification of Radiolabeled Oligonucleotides by Precipitation with Cetylpyridinium Bromide Protocol]

Category: Nucleic Acid Electrophoresis Protocols: DNA Electrophoresis Protocols: DNA Electrophoresis in Agarose Gels Protocols

DNA fragments separated by electrophoresis through gels cast with low-melting-temperature agarose are recovered by melting the agarose and extracting the resulting solution with phenol:chloroform.  The protocol works best for DNA fragments ranging in size from 0.5 kb to 5 kb. - [Read Recovery of DNA from Low-melting-temperature Agarose Gels: Organic Extraction Protocol]

Category: Viral Manipulation Protocols: M13 Phage Manipulation Protocols

Technique yields a heterogeneous population of short radiolabeled molecules 200-300 nucleotides in length. These probes are synthesized, as in Synthesis of Single-stranded DNA Probes of Defined Length from Bacteriophage M13 Templates, by extension of an oligonucleotide primer on a single-stranded DNA template. The radiolabeled products of the reaction are then separated from the template by electrophoresis through a denaturing gel from which they are eluted directly into hybridization buffer. - [Read Synthesis of Single-stranded DNA Probes of Heterogeneous Length from Bacteriophage M13 Templates]

Category: Immunology: Chemotaxis

TransWell Chemotaxis protocol. trans-well chemotaxis method from bioprotocol. The protocol is a method for studying migration towards a concentration gradient of chemoattractant of leukocytes (neutrophils, monocytes and lymphocytes) or other migratory cells. An upper chamber containing a suspension of cells is separated by a membrane from a lower chamber containing medium with chemoattractant. Chemotaxis of the cells from the upper chamber into the lower chamber can be quantified. - [Read TransWell Chemotaxis Protocol.]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Method is used to assess (roughly) the integrity of total RNA samples by visualization of discreet 18S and 28S ribosomal RNAs.  Total RNA is separated by electrophoresis through a 1% agarose gel containing 1.3 ìM ethidium bromide.  Binding of the ethidium bromide to the RNA allows visualization of the separated RNA molecules when the gel is exposed to ultraviolet (UV) light. - [Read Visualization of RNA Preparations on 1% Agarose Gels Protocol]

Category: Developmental Biology: Embryology Protocols

This protocol describes a method for visualizing early embryo implantation sites using Chicago Sky Blue 6B dye. Once implantation and interimplantation sites are identified and separated, they can be used for cellular, biochemical, and molecular biology analyses. - [Read Visualizing Early Embryo Implantation Sites by Dye Injection Protocol]