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Processing of Microdissected Tissue for Molecular Analysis Proteomics-based Studies - http://cgap-mf.nih.gov/Protocols/Processing/Proteomics.html

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

The methodology and buffers utilized for processing microdissected samples for protein-based studies is variable depending on the downstream molecular analysis method. - [Read Processing of Microdissected Tissue for Molecular Analysis Proteomics-based Studies]

Protocol for Cycling Tubulin - http://www.ciwemb.edu/labs/koshland/Protocols/MICROTUBULE/cyclingmicrotub.html

Category: Cell Biology and Cell Culture

Protocol for cycling tubulin using the buffers Glycerol PB and BRB80. - [Read Protocol for Cycling Tubulin]

Protocol for Primary Cultures of HUVECs - http://www.huvec.com/documents/Protocol-HUVECs.pdf

Category: Cell Biology and Cell Culture: Endothelial Cell Protocols

Protocol for primary cultures of HUVECs. Includes: Buffers and reagents (Fetal calf serum, Phosphate Buffer Saline (PBS), Collagenase, Buffer for conservation and transport of umbilical cords, Culture medium); Cell culture. - [Read Protocol for Primary Cultures of HUVECs]

Protocol: Luciferase assay protocol - http://medicine.ucsd.edu/hypertension/list_of_protocols/5_luciferase_assay.htm

Category: Luciferase and Dual Luciferase Assays

Protocol: Luciferase assay protocol. Simple Recipies for Luciferase assay buffers I and II. - [Read Protocol: Luciferase assay protocol]

Purification of DNA Recovered Anion-exchange Chromatography Protocol - http://cshprotocols.cshlp.org/cgi/content/extract/2006/2/pdb.prot4024

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for the purification of DNA recovered from agarose and polyacrylamide gels by anion-exchange chromatography. Fragments of DNA recovered from agarose gels are sometimes poor templates or substrates in subsequent enzymatic reactions. This problem can be solved by binding the DNA to a positively charged matrix, such as DEAE-Sephadex or DEAE-Sephacel, in buffers of low ionic strength. After washing the matrix, the DNA is eluted by raising the strength of the buffer. - [Read Purification of DNA Recovered Anion-exchange Chromatography Protocol]

Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3655

Category: Chromatography Protocols: Affinity Chromatography Protocols

Recombinant proteins, constructed in pGEX vectors, are fused to glutathione S-transferase (GST) and can be purified to near homogeneity by affinity chromatography on glutathione-agarose. Bound GST-fusion proteins are readily displaced from the column by elution with buffers containing free glutathione. - [Read Purification of Fusion Proteins by Affinity Chromatography on Glutathione Agarose Protocol]

Restriction Digestion Methods - http://hg.wustl.edu/hdk_lab_manual/rdm/rdmcontents.html

Category: Molecular Biology Protocols: Restriction Enzyme Digestion

Restriction Digestion Methods. Donis-Kellor lab manual. Restriction Enzyme Digests, Restriction Enzyme Buffers, Restriction Digestion of Plasmid, Cosmid, and Phage DNAs. - [Read Restriction Digestion Methods]

Separating Proteins Using Ion-Exchange Chromatography Protocol - http://cshprotocols.cshlp.org/cgi/content/extract/2006/1/pdb.prot4196

Category: Chromatography Protocols: Ion Exchange Chromatography Protocols

Protocol for separating proteins using ion-exchange chromatography. Protocol details the practical considerations of an ion-exchange chromatography (IEC) experiment. The choice of what type of ion exchanger to use, as well as the composition of the buffers used in this experiment, should be determined prior to beginning this protocol. - [Read Separating Proteins Using Ion-Exchange Chromatography Protocol]

Single-tube Coupled Transcription/Translation Reactions for Eukaryotic In Vitro - http://www.promega.com/tbs/tm045/tm045.pdf

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]

Southern Hybridization of Radiolabeled Probes to Nucleic Acids Immobilized on Membranes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4044

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]

Transfer and Fixation of Denatured RNA to Membranes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot4060

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]

Western Blot Protocol - http://genetics.med.harvard.edu/~cepko/protocol/mike/W1.html

Category: Western Blot Protocols: General Western Blot Methods Protocols

This protocol was developed for the BIORAD protein gel and transfer apparatus. The buffers can be used with any electrophoresis/transfer system. - [Read Western Blot Protocol]

Yeast Chromatin Immunoprecipitation (ChIP) - http://www.fhcrc.org/science/labs/hahn/methods/mol_bio_meth/hahnlab_ChIP_method.html

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Chromatin IP Method, ChIP buffers, Notes on ChIP Method, Quantative PCR using 32P dCTP. Hahn Lab. - [Read Yeast Chromatin Immunoprecipitation (ChIP)]

Yeast Immunofluorescence protocol - http://clone.concordia.ca/yeast2/protocols/Immunofluorescence_protocol.pdf

Category: Molecular Imaging: Immunofluorescence Microscopy

Yeast Immunofluorescence protocol, includes recipes for Immunofluorescence Buffers. Concordia. - [Read Yeast Immunofluorescence protocol]

Yeast Nuclear Extract (Small Scale) - http://www.fhcrc.org/science/labs/hahn/methods/biochem_meth/polii_nuc_ext.html

Category: Protein Protocols: Nuclear Extraction Protocols

Nuclear Extract preparation protocols, Buffers and solutions for nuclear extracts. Hahn Lab - [Read Yeast Nuclear Extract (Small Scale)]

Articles (1-10 of 16)

Single Stranded Plasmid DNA Isolation Protocol

A Single Stranded Plasmid DNA Isolation Protocol describing the production and isolation of single-stranded DNA (ssDNA) using bacteriophagemid-containing bacteria and helper phage. Infection of the host cells with helper phage allows for packaging of ssDNA into bacteriophage. The ssDNA can then be isolated from phage particles.

Genomic DNA Labeling of Microarrays Protocol

DNA microarrays are an ordered arrangement of DNA molecules complementary to genes of interest that are "spotted" by robotic equipment onto a glass slide substrate. The expression of genes in cells can be monitored with microarrays by preparing cDNA from the mRNA of cells of interest and measuring the hybridization to the microarray. This protocol describes the labeling of genomic DNA for use as a probe for hybridization to the cDNA spotted on the array.

Drosophila Cell Transfection Protocol

A simple Drosophila tissue culture cell transfection method.

Tubulin Polymerization Protocol using GTP and GMPCPP

Tubulin is polymerized into microtubules by incubating tubulin at 37°C with GTP. A nucleation seed is added when the purpose is to assay microtubule elongation. Tubulin can also be polymerized for the purposes of recycling the tubulin or labeling the microtubules with fluorescently labeled tubulin. Based on the protocol by Timothy Mitchison of Harvard University.

Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA

This Microarray Protocol Preparation of Fluorescent DNA Probes from Human mRNA protocol describes the production of probes labeled with the fluorescent dyes, Cy3 and Cy5, following the synthesis of cDNA from human mRNA and the hybridization of the probes to DNA microarrays.

Acid Guanidinium Thiocyanate RNA Isolation Phenol Chloroform Extraction

A single step RNA isolation protocol using Phenol Chloroform Extraction and Acid Guanidinium Thiocyanate. This RNA isolation method uses the fact that guanidinium thiocyanate can simultaneously lyse the cells and inactive cellular RNAses during the initial RNA isolation step allow a single step in the method.

Acid Washing Microinjection Pipettes Protocol

Acid Washing of Glass microinjection pipettes protocol.

In Vitro Translated Xenopus Mos Kinase Assay Protocol

In Vitro Translated Xenopus Mos Kinase Assay Protocol. In response to progesterone, immature Xenopus oocytes mature to eggs that can be fertilized. The Mos protein kinase is essential for oocyte maturation, most likely due to its ability to activate the MAP kinase cascade. This MAP kinase cascade eventually leads to the activation of Cdc2/cyclin B and entry into M phase. In this protocol, tagged Mos kinase is translated in vitro, immunopurified, and used in a kinase assay.

Immobilized Antigen Phage Antibody Selection Protocol

A protocol for the selection of Phage Antibodies using Immobilized Antigen. This method describes the selection of antibodies from bacteriophage antibody libraries that recognize a specific antigen. The phage display library of antibody-displaying phage particles is exposed to antigen attached to a solid substrate (Nunc Immuno™ tubes). The phage particles with affinity for antigen bind to the immobilized antigen and are selected from the library of phage expressing antibodies.

Absorption Spectroscopy and Quantification of Filamentous Phage Protocol

Unlike spherical phage, such as T4 and λ, which have roughly equal weight ratios of protein to DNA, filamentous phage have about six times more protein than DNA; the protein therefore contributes substantially to the absorption spectrum.

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