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Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4659

Category: Microinjection Protocols: Microinjection of Proteins Protocols

Activation and inactivation of proteins using photoactivation of caged peptides or proteins offer insights into cellular dynamics not achievable using genetic means. The ability to selectively alter the activity of a specific protein at a defined time and location inside a cell allows the correlation of changes in protein activity and cellular behavior. A caged compound, peptide, or protein is prepared by covalently linking it to a photolabile, protecting group. - [Read Introduction of Caged Peptide/Protein into Cells Using Microinjection Protocol]

Isolation of proteins from inclusion bodies - http://www.its.caltech.edu/%7Ebjorker/Isolation%20of%20proteins%20from%20.pdf

Category: Recombinant Protein Protocols

Isolation of proteins from inclusion bodies Protcol. Expression of recombinant proteins. Dissolving of recombinant protein. Refolding buffer. BJÃ–RKman group - [Read Isolation of proteins from inclusion bodies]

Lab FAQS: Working with Proteins. - http://www.roche-applied-science.com/labfaqs/p3_1.htm

Category: Protein Protocols

Lab FAQS: Working with Proteins. - [Read Lab FAQS: Working with Proteins.]

Large scale nuclear extract preparation - http://mesentsev.narod.ru/Biblioteque/Hattori.html

Category: Protein Protocols: Nuclear Extraction Protocols

Homogenize cells or tissue, Pellet nuclei, Lyse nuclei, Reprecipitate nuclear proteins, dialyze nuclear extract. Hattori M et al., DNA Cell Biol. 1990. Homogenization buffer and dialysis buffer recipes. - [Read Large scale nuclear extract preparation]

Live-Cell Imaging of GFP in Plants - http://www.cshprotocols.org/cgi/content/full/2007/3/pdb.ip31

Category: Microscopy Protocols: Live-Cell Imaging Protocols

GFP serves as a molecular marker that can be imaged dynamically in living cells, both in its native form & as a fusion to other proteins. For GFP imaging, plants present the challenge of autofluorescence from chlorophyll, lignified cell walls, vacuolar contents, and other cell materials, all of which can obscure the GFP signal. Maximizing the signal-to-noise ratio is a major concern, and careful consideration should be given to the choice of tissue imaged, GFP expression level, etc. - [Read Live-Cell Imaging of GFP in Plants]

Lysing Tissue-Culture Cells for Immunoprecipitation Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4531

Category: Cell Culture Protocols: Cell Lysis Protocols

For cells grown in tissue culture, the most useful method of lysis is treating with detergents, as described in this protocol. Non-ionic detergents, such as NP-40, solubilize the plasma and intracellular membranes, break many weak intermolecular bonds, and solubilize most of the commonly studied protein antigens. RIPA lysis buffer may be used as a more rigorous extraction buffer to release all but the insoluble proteins of the cell and to break most weak noncovalent interactions. - [Read Lysing Tissue-Culture Cells for Immunoprecipitation Protocol]

Magnetic techniques for the isolation and purification of proteins and peptides - http://www.biomagres.com/content/2/1/7

Category: RNA Protocols: RNA-binding Protein Purification

Magnetic techniques for the isolation and purification of proteins and peptides. Safarik et al. 2004 - [Read Magnetic techniques for the isolation and purification of proteins and peptides]

MALDI Matrices - http://www.narrador.embl-heidelberg.de/GroupPages/PageLink/activities/protocols/maldimatrices.html

Category: Mass Spectrometry Protocols

MALDI Matrices. Commonly used MALDI matrices for analysis of peptides, proteins, carbohydrates, and nucleic acids using 337 nm or 355 nm UV lasers. All matrices can be used for sample preparation using the Dried Droplet Method whereas only matrices solubl - [Read MALDI Matrices]

Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3947

Category: DNA Protein Interactions Protocols: Dnase Footprinting Assay Protocols

Dnase I is used to fragment a radiolabeled target DNA in the presence and absence of a nuclear extract. A "footprint" is generated when a protein binds to the target and protects a specific segment of DNA from the nucleolytic activity of Dnase I. By comparing the electrophoretic mobility of the Dnase I cleavage products to those of a sequence ladder derived from the same DNA fragment, the position(s) of the DNA sequences recognized by DNA-binding proteins can be determined. - [Read Mapping Protein-binding Sites on DNA by Dnase I Footprinting Protocol]

Mass spectrometry analysis and identification of proteins - http://www.healthsystem.virginia.edu/internet/biomolec/mass_spectrometry.cfm

Category: Mass Spectrometry Protocols

Protein sequencing by mass spectrometry. Mass analysis of peptides and proteins. Identification of modifications in proteins. Good information.Univ. Virginia. Dr. Nicholas Sherman - [Read Mass spectrometry analysis and identification of proteins]

Measurement of Cell Adhesion Under Static Conditions - http://www.glycotech.com/protocols/Proto8.html

Category: Cell Biology and Cell Culture: Cell Adhesion Protocols

Many proteins and molecules promote cell adhesion including several cell surface carbohydrate binding proteins. Cell adhesion measurements on 96-well microtiter plate format are difficult due to the shear forces generated by washing the wells. The protocol here introduces the use of a liquid-filled wash chamber that separates unbound cells by gravity. This eliminates uncontrolled shear forces and passage of adherent cells through a liquid/air interface. John L. Magnani~GlycoTech Corporation. - [Read Measurement of Cell Adhesion Under Static Conditions]

Mechanical Tracing of Protein Function in the Drosophila Ear Protocol - http://www.natureprotocols.com/2006/11/03/mechanical_tracing_of_protein.php

Category: Drosophila Protocols

Method relies on the examination of the ear’s mechanics, which is actively modulated by the motility of auditory neurons and reflects the function of mechanosensory proteins these cells comprise [5-7]. Mechanical signatures arising from the motility of the neurons are assayed by measuring the vibrations of the antennal sound receiver in the presence and absence of sound. - [Read Mechanical Tracing of Protein Function in the Drosophila Ear Protocol]

Metabolic Labeling of Plant Cell Cultures with K15NO3 Protocol - http://www.plantmethods.com/content/2/1/14

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

Protocol for metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites. - [Read Metabolic Labeling of Plant Cell Cultures with K15NO3 Protocol]

Methanol-Chloroform Precipitation of Proteins - http://www.healthsystem.virginia.edu/internet/biomolec/methanolchloroform.cfm

Category: Mass Spectrometry Protocols

Methanol-Chloroform Precipitation of Proteins. A procedure for precipitating proteins from solution, including detergent solutions, is from Wessel and FlÃ¼gge (1984). Univ. Virginia. - [Read Methanol-Chloroform Precipitation of Proteins]

Microinjection of Protein Samples Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4657

Category: Microinjection Protocols: Microinjection of Proteins Protocols

This protocol describes a method for microinjecting proteins into the nucleus or cytoplasm of adherent cells. Microinjection equipment can be obtained from a number of suppliers; this protocol has been used with the Narishige IM-200 air pressure regulator and the Leitz micromanipulator. Using this system, it is possible to microinject a constant volume within a 50% difference among cells. - [Read Microinjection of Protein Samples Protocol]

Nondenaturing Protein Immunoprecipitation from Mammalian Cells Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4620

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol describes a nondenaturing immunoprecipitation (IP) for mammalian cells. Prefer to use denaturing IPs to recover labeled proteins from pulse-chase experiments. However, the nondenaturing protocol is useful when one wishes to separate soluble from insoluble proteins, or when the antibody being used recognizes a native epitope. - [Read Nondenaturing Protein Immunoprecipitation from Mammalian Cells Protocol]

Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4210

Category: Chromatography Protocols: Dye Ligand Affinity Chromatography Protocols

Protocol for the optimization of absorption condition for dye-ligand affinity chromotography. Generally, low pH and low ionic strength, absence of phosphate ions, and the presence of divalent metals ions increase the binding of proteins to immobilized triazine dyes. - [Read Optimization of Adsorption Conditions for Dye-Ligand Affinity Chromatography Protocol]

Optimization of Imidazole Concentrations for Immobilized Metal-Ion Affinity Chromatography Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/9/pdb.prot4261

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for the optimization of imidazole concentrations for immobilized metal-ion affinity chromatography. Most samples from which histidine-tagged proteins are to be purified also contain endogenous protein contaminants that bind to the immobilized metal-ion affinity chromatography (IMAC) adsorbent. Usually, these proteins bind more weakly than the histidine-tagged protein. - [Read Optimization of Imidazole Concentrations for Immobilized Metal-Ion Affinity Chromatography Protocol]

Overview of Affinity Purification in Combination with Mass Spectrometry Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/30/pdb.top3

Category: Chromatography Protocols: Affinity Chromatography Protocols

Protein complexes can be isolated by several different approaches. For example, a protein can be tagged with an epitope such as Flag or TAP and then overexpressed in a target cell, allowing the interacting proteins to be purified. Similarly, epitope tags can be homologously recombined into the endogenous locus ("knocked-in"), allowing protein complexes containing the tagged proteins to be isolated at their natural expression level. - [Read Overview of Affinity Purification in Combination with Mass Spectrometry Protocol]

Phosphoamino acid analysis of non-TCA precipitable proteins - http://kinase.uhnres.utoronto.ca/methods/PAA.html

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Phosphoamino acid analysis of non-TCA precipitable proteins and Alkali treatment of gels protocols. Woodgett Lab. - [Read Phosphoamino acid analysis of non-TCA precipitable proteins]

Preparation and Loading of Protein Samples for Microinjection Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4656

Category: Microinjection Protocols: Microinjection of Proteins Protocols

his protocol provides methods for the preparation of protein samples and for loading them into pulled microinjection pipettes. Stock solutions of proteins are thawed, diluted (if desired), centrifuged at high speed to remove aggregates, and kept on ice until loading. Loading into micropipettes can be done using either a "front-loading" or a "backfilling" procedure. - [Read Preparation and Loading of Protein Samples for Microinjection Protocol]

Preparation of a Prepacked IMAC Column Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/1/pdb.prot4214

Category: Chromatography Protocols: Immobilized Metal Ion Affinity Chromatography Protocols

Protocol for the preparation of a prepacked IMAC column. Purification of histidine-tagged proteins on a milligram scale can be performed conveniently on a small (1to 5-ml) immobilized metal-ion chromatography (IMAC) column using a syringe to load the sample, wash the column, and elute the protein. - [Read Preparation of a Prepacked IMAC Column Protocol]

Preparation of Arabidopsis Chloroplasts Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4681

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

It is desirable to prepare subcellular fractions, either to localize proteins or to improve the sensitivity of protein detection. This procedure describes the enrichment of chloroplasts from Arabidopsis. - [Read Preparation of Arabidopsis Chloroplasts Protocol]

Preparation of Arabidopsis Mitochondria Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/1/pdb.prot4682

Category: Plant Biology Protocols: Arabidopsis Tissue Culture

It is often desirable to prepare subcellular fractions, either to localize proteins or to improve the sensitivity of protein detection. This procedure describes the enrichment of mitochondria from Arabidopsis. - [Read Preparation of Arabidopsis Mitochondria Protocol]

Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3769

Category: Viral Culture and Isolation Protocols: Viral Culture Protocols

In this protocol, a bacterial lysogen is constructed from a recombinant bacteriophage {lambda} encoding a fusion protein of interest. The resulting lysogenic colonies are induced to synthesize the fusion protein, which is then isolated in preparation for functional and biochemical analyses. - [Read Preparation of Lysates Containing Fusion Proteins Encoded by Bacteriophage {lambda} Lysogens]

Articles (1-3 of 3)

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.

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.