membranes Protocols

Category: RNA Protocols: cDNA Libraries Library

Denaturation Solution, Neutralization Solution, Rinse Solution, Crosslink DNA to membranes using Stratalinker UV crosslinker. Donelson Lab, Univ. Iowa. - [Read Nitrocellulose Membrane Washing Procedure for cDNA Screening]

Category: DNA Protocols: PCR Polymerase Chain Reaction Protocols

PCR protocol for cDNA Arrays on Membranes. PDF file - [Read PCR protocol for cDNA Arrays on Membranes. PDF file.]

Category: PCR Protocols

PCR protocol for cDNA Arrays on Membranes. PDF file - [Read PCR protocol for cDNA Arrays on Membranes. PDF file.]

Category: Antibody Protocols: Antibody Production Protocols

Polyclonal antibodies can be isolated from animal plasma or serum using the procedure described in this protocol. The Gradiflow BF400 instrument has two liquid streams that circulate through a separation cartridge positioned between two electrodes and composed of three hydrogel polyacrylamide membranes, which define the channels for the two sample streams. The central membrane forms a physical barrier between the two streams. - [Read Preparation of Polyclonal Antibodies from Plasma or Serum Using the Gradiflow BF400]

Category: Fungi Protocols: Neurospora Protocols

Protocol for Preparing Vacuolar Membranes And Other Membrane Fractions from Neurospora Crassa (4 liter prep, easily scaled up to 10, 12, 24, or 30 l). - [Read Preparing Vacuolar Membranes And Other Membrane Fractions from Neurospora Crassa Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols: Apoptosis Cytometry Protocols

Propidium iodide (PI) intercalates into double-stranded nucleic acids. It is excluded by viable cells but can penetrate cell membranes of dying or dead cells. - [Read Propidium Iodide Staining of Dead Cells for Flow Cytometry Protocol]

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

Protocol includes information on: Preparation of Membranes; One-Stage Reaction; Two-Stage Reaction using Normal Amount of Membranes; Two-Stage Reaction using Low Concentration of Membranes. - [Read Protcol for In Vitro Endoplasmic Reticulum to Golgi Transport Reaction in Yeast]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

The first part of the isolation procedure is a flotation through a continuous iodixanol gradient; this gradient is essentially a resolving gradient in which the caveolin-rich vesicles are concentrated in the top third of the gradient, while the predominantly caveolin-poor vesicles band in denser regions. A second discontinuous gradient is essentially a concentration gradient to band the caveolin-rich vesicles sharply at an interface. - [Read Purification of Caveolae Membranes from a Plasma Membrane Fraction of Cultured Cells and Tissues]

Category: Cell Organelle Protocols: Subcellular Fractionation Protocols: Membrane Isolation Protocols

This protocol uses a "light mitochondrial" pellet from a mammalian liver homogenate. The gradient thus has to resolve a variety of denser components (peroxisomes, lysosomes, mitochondria) from the Golgi membranes, which have a low density in iodixanol (1.06-1.09 g/ml) [1]. The protocol is specifically tailored to the purification of Golgi membranes from this pellet and is unsuitable for the isolation or analysis of other organelles present in the light mitochondrial fraction. - [Read Purification of Golgi Membranes from a Light Mitochondrial Fraction in a Self-Generated Gradient]

Category: Nucleic Acid Modification Protocols: Random Primer Labeling Protocols

Protocol for random primer labeling of poly A and RNA. Includes: Random primer reaction; cDNA purification; Hybridization; Washing; Stripping of membranes. - [Read Random Primer Labeling of PolyA and RNA Protocol]

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

Recovery of bands of DNA from agarose gels by electrophoresis onto a sliver of DEAE-cellulose membrane can be performed simultaneously on many samples and reliably gives high yields of fragments between 500 bp and 5 kb in length. - [Read Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes Protocol]

Category: Pharmacology and Toxicology Protocols: Cytotoxicity Protocols: Cell Specific Cytotoxicity Assays Protocols

An in vitro red blood cell assay is presented which allows the estimation of the irritation potential of tensides and tenside containing materials such as shampoos, shower gels, cleaning products, etc. The estimation is based on the fact that surfactants interact strongly with cellular membranes and proteins.  Both effects are measured photometrically by use of the inherent native dye, oxyhemoglobin. - [Read Red Blood Cell Test System Protocol]

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

Information for protocol using single-tube, coupled transcription/translation reactions for eukaryotic in vitro translation. Includes information on: Translation Procedure; Positive Control Translation Reactions Using Luciferase; Cotranslational Processing Using Canine Pancreatic Microsomal Membranes; Post-Translational Analysis; Positive Control Luciferase Assays; Composition of Buffers and Solutions; Luciferase SP6/T7 Control DNAs - [Read Single-tube Coupled Transcription/Translation Reactions for Eukaryotic In Vitro]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol describes the first stages of Southern blotting: digestion of genomic DNA with one or more restriction enzymes, separation of the resulting fragments by electrophoresis through an agarose gel, and transfer of the denatured fragments to a membrane by downward capillary transfer. - [Read Southern Blotting: Capillary Transfer of DNA to Membranes Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for southern blotting: simultaneous transfer of DNA from a single agarose gel to two membranes. DNA can be simultaneously transferred from opposite sides of a single agarose gel to two membranes.  Bidirectional transfer occurs rapidly at first, but soon slows down as the gel becomes dehydrated.  Because the efficiency of transfer is low, the method works best when the target sequences are present in high concentration - [Read Southern Blotting: Simultaneous Transfer of DNA from a Single Agarose Gel to Two Membranes Protocol]

Category: Nucleic Acid Detection Protocols: Nucleic Acid Blotting Protocols

Protocol for southern hybridization of radiolabeled probes to nucleic acids immobilized on membranes. Protocol describes how to carry out Southern hybridizations at high stringency in phosphate-SDS buffers.  Although a wide variety of formats are available, most Southern hybridizations are carried out in heat-sealable bags, roller bottles, or plastic boxes. - [Read Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes Protocol]

Category: Nucleic Acid Electrophoresis Protocols: RNA Electrophoresis Protocols

Protocol describes the transfer of RNA from agarose gels to neutral or positively charged nylon membranes, using upward capillary flow of neutral or alkaline buffers.  RNA becomes covalently fixed to positively charged nylon membranes during transfer in alkaline buffers.  However, treatment by UV irradiation or heating is required to fix RNA to neutral membranes. - [Read Transfer and Fixation of Denatured RNA to Membranes Protocol]

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

To transfer phage DNA to nylon membranes so that a phage library can be screened by hybridization. - [Read Transfer of Phage DNA from Plaques to Nylon Membrane or Nitrocellulose Protocol]

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Use of the chemiluminescence-producing alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'-tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD, also known as adamantyl-1,2-dioxetane phosphate), or its dioxetane relatives provides a substantial increase in sensitivity over colorimetric substrates and radiochemical methods currently used for the detection of antigen-antibody complexes immobilized on nylon or PVDF membranes. - [Read Western Analysis Using the Chemiluminescent Alkaline Phosphatase Substrate CSPD Protocol]

Category: Protein Protocols: Western Blot Protocol: Troubleshooting Western Blots

Streaking of Western Blots, Weak Bands or Weak Staining of Western Blots, Poor Transfer of Membranes, Dirty Blot, Multiple Bands on Western Blot Membrane and Film. Western Blot Info. - [Read Western Blot Troubleshooting]

Category: Yeast Protocols

Plasma membranes are isolated from the yeast Saccharomyces cerevisiae. The cell wall is initially digested by helicase, followed by hypoosmotic lysis and homogenization. Membranes are prepared by subsequent differential centrifugation. The activity of the H+-ATPase is then determined by measuring the amount of inorganic phosphate released from ATP. - [Read Yeast Plasma Membrane H+ -ATPASE Toxcity Test Protocol]