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Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/2/pdb.prot3785

Category: Viral Manipulation Protocols: Phage Lamda Manipulation Protocols

Protocol describes how to identify cloned cDNAs encoding proteins that bind to specific DNA sequences. The methods used are very similar to those used for immunological screening of expression libraries except that the nitrocellulose filters carrying immobilized proteins are screened with 32P-labeled double-stranded DNA rather than with antibodies. - [Read Identifying DNA-binding Proteins in Bacteriophage ambda Expression Libraries Protocol]

Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4673

Category: Reporter Gene Assays: GFP Assay Protocols

Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. These methods should be followed in the order presented, but any of them can be omitted when not needed. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle - http://www.cshprotocols.org/cgi/content/abstract/2007/3/pdb.prot4673

Category: Microinjection Protocols

Fluorescence microscopy provides a powerful tool for imaging molecular components in living cells. Specific molecular components can be efficiently labeled by a combination of three methods: chemical transfection of GFP-fusion constructs, staining of chromosomes with the DNA-specific, fluorescent dye Hoechst 33342, and microinjection of fluorescently conjugated proteins. This procedure provides an example of using all three methods in sequence to label components of living HeLa cells. - [Read Imaging Hoechst-Labeled Chromosomes and Fluorescent Proteins during the Cell Cycle]

Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells - http://www.cshprotocols.org/cgi/content/abstract/2006/29/pdb.prot4603

Category: Microscopy Protocols: Live-Cell Imaging Protocols

Using confocal laser-scanning microscope & GFP fusion proteins in time-lapse imaging to visualize the behavior of organelles and to track membrane-bound transport intermediates that bud off from organelles. Practical issues related to construction & expression of GFP fusion proteins are discussed. Essential for optimizing the brightness and expression levels of GFP fusion proteins so that intracellular membrane-bound structures containing these fusion proteins can be readily visualized. - [Read Imaging of Organelle Membrane Systems and Membrane Traffic in Living Cells]

Immunoblotting: Antigen Detection Using Chemiluminescence Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4271

Category: Western Blot Protocols: Antigen Detection Methods Protocols

The blot is blocked to prevent nonspecific adsorption of the immunological reagents. Antibodies are then bound to the proteins immobilized on the membrane, and the antigen is detected by labeling the antibodies with conveniently identified tags. Common labeling methods for chemiluminescent detection include anti-immunoglobulin antibody-coupled enzymes such as horseradish peroxidase, which catalyzes the oxidation of luminol and in turn releases light. - [Read Immunoblotting: Antigen Detection Using Chemiluminescence Protocol]

Immunoblotting: Antigen Detection Using Chromogenic Methods Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4272

Category: Western Blot Protocols: Antigen Detection Methods Protocols

Immunoblotting: Preparing Immunoprecipitated Proteins Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4300

Category: Western Blot Protocols

Protein immunoprecipitation can be a useful preparative step for immunoblotting. For very rare proteins, the protein of interest can be purified and concentrated by standard immunoprecipitation techniques before immunoblotting. In addition, protein-protein interactions can be tested with an immunoprecipitating antibody that is specific for one protein of a complex and an immunoblotting antibody that is specific for a second member of the complex. - [Read Immunoblotting: Preparing Immunoprecipitated Proteins Protocol]

Immunoblotting: Preparing Protein Solutions Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/21/pdb.prot4298

Category: Western Blot Protocols: General Western Blot Methods Protocols

Protocol describes how proteins are mixed with a sample buffer and prepared for electrophoresis on standard Tris/glycine SDS-polyacrylamide gels. - [Read Immunoblotting: Preparing Protein Solutions Protocol]

Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4301

Category: Western Blot Protocols: General Western Blot Methods Protocols

The transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using a semi-dry method is achieved by placing the gel next to a piece of nitrocellulose filter. This sandwich is placed directly between two plate electrodes, and the proteins are then transferred from the gel onto the filter. - [Read Immunoblotting: Semi-Dry Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]

Immunoblotting: Submerged Electrophoretic Transfer of Proteins from Gels to Membranes Protocol - http://www.cshprotocols.org/cgi/content/extract/2006/16/pdb.prot4302

Category: Western Blot Protocols: General Western Blot Methods Protocols

Transfer of proteins from a Tris/glycine SDS-polyacrylamide gel to a membrane using the submerged method is achieved by placing the gel next to a piece of nitrocellulose filter, submerging this sandwich in a large volume of transfer buffer in a transfer tank, and running current from one side of the transfer tank to another. The proteins are then eluted by transferring them from the gel onto the filter. - [Read Immunoblotting: Submerged Electrophoretic Transfer of Proteins from Gels to Membranes Protocol]

Immunodepletion Protocol - http://www.fgsc.net/neurosporaprotocols/Immunodepletion%20final.pdf

Category: Protein Protocols

Protocol for studying quantitative relationships of proteins in a complex. - [Read Immunodepletion Protocol]

Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/14/pdb.prot4779

Category: Immunohistochemistry Protocols: Cryosectioning Protocols

This protocol describes methods for the fixation, cryosectioning, and immunohistochemical detection of proteins in embryonic and adult zebrafish eyes. The methods described can easily be adapted for use on other zebrafish tissues. - [Read Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol]

Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol - http://www.cshprotocols.org/cgi/content/abstract/2007/14/pdb.prot4779

Category: Laboratory Model Organisms Protocols

Protocol describes methods for the fixation, cryosectioning, and immunohistochemical detection of proteins in embryonic and adult zebrafish eyes. The methods described can easily be adapted for use on other zebrafish tissues. - [Read Immunohistochemistry on Cryosections from Embryonic and Adult Zebrafish Eyes Protocol]

Immunoprecipitation of Chromatin from Fixed Yeast Protocol PDF - http://derisilab.ucsf.edu/pdfs/BeadBeatIP_Protocol.pdf

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Chromatin-IP (or ChIP) has been a useful method for the localization of proteins to specific regions of DNA. ChIP applied to microarrays (ChIP on Chip) Brown Lab - [Read Immunoprecipitation of Chromatin from Fixed Yeast Protocol PDF]

Immunoprecipitation of Epitope-Tagged Proteins from Yeast Protocol - http://www.med.unc.edu/~hdohlman/TAP.html

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

Protocol for immunoprecipitation of epitope-tagged proteins from yeast. - [Read Immunoprecipitation of Epitope-Tagged Proteins from Yeast Protocol]

Immunoprecipitation of Proteins Method - IP protocol - http://www.upstate.com/misc/protocols.q.prot.e.immuno/Immunoprecipitation+of+Proteins

Category: Protein Protocols: Immunoprecipitation Protocols

Ip Protocol using protein A or G beads. Upstate. - [Read Immunoprecipitation of Proteins Method - IP protocol]

Immunoprecipitation TroubleShooting Guide Sigma - http://www.sigmaaldrich.com/img/assets/8181/ImmunoprecipTroubleshoot.pdf

Category: Protein Protocols: Immunoprecipitation Protocols: Immunoprecipitation Troubleshooting

Immunoprecipitation TroubleShooting Guide. Sigma Aldrich. Problems: No binding, High background or unwanted proteins precipitate. With Solutions. - [Read Immunoprecipitation TroubleShooting Guide Sigma]

Immunoprecipitation: Preclearing the Lysate Protocol - http://www.cshprotocols.org/cgi/content/abstract/2006/23/pdb.prot4535

Category: Immunology: Immunoprecipitation Protocols: Protein Immunoprecipitation Protocols

To reduce backgrounds and to improve the signal-to-noise ratio, an antibody that does not recognize the antigen being studied can be added to the lysate and processed as for a normal immunoprecipitation. Any nonspecific proteins that might contaminate the final immunoprecipitation step are presumably removed with this irrelevant antibody. - [Read Immunoprecipitation: Preclearing the Lysate Protocol]

Immunostaining of DDR Proteins in Senescent Cells Protocol - http://www.natureprotocols.com/2006/12/06/immunostaining_of_ddr_proteins_1.php

Category: Immunology: Immunofluorescence Protocols: Immunofluorescence Staining Protocols

Protocol for immunostaining of DDR proteins in senescent cells. Detailed methodology to study the activation of a number of DNA damage response factors in senescent human fibroblasts. - [Read Immunostaining of DDR Proteins in Senescent Cells Protocol]

Immunostaining Thin Layer Chromatograms Of Glycolipids - http://www.glycotech.com/protocols/Proto5.html

Category: Molecular Biology Protocols: Carbohydrate Protocols

Immunostaining thin layer chromatograms TLC is a very sensitive detection technique of functionally active carbohydrate ligands of protein receptors. Carbohydrate structures are detected in glycolipids from complex mixtures of molecules extracted from the relevant target tissue. Proteins analyzed can be antibodies, chimeric Ig proteins, selectins, lectins, toxins, and other carbohydrate binding proteins. John L. Magnani~GlycoTech Corporation, Rockville, Maryland - [Read Immunostaining Thin Layer Chromatograms Of Glycolipids]

Import Assays Into Chloroplasts: Integration or Transport into Thylakoids - http://www.hos.ufl.edu/clineweb/Protocols/ProteinImport.htm

Category: Protein Protocols: Protein Trafficking

Import assays into chloroplasts. Includes: Preparation of chloroplasts and lysates; Transport of lumenal proteins. - [Read Import Assays Into Chloroplasts: Integration or Transport into Thylakoids]

In-gel digestion of proteins for peptide fingerprint mapping - http://www.bio.vu.nl/vakgroepen/mnb/proteomics/6.in-geldigestion.html

Category: Mass Spectrometry Protocols

Peptide Mapping Analysis Techniques. Dr.Aebersold, Vrije Universiteit Amsterdam - [Read In-gel digestion of proteins for peptide fingerprint mapping]

In-Gel Digestion of Proteins Separated by Polyacrylamide Gel Electrophoresis - http://web.archive.org/web/20010906052645/http://www.pharma.ethz.ch/bmm/protocols/ingeldig.html

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]

In-gel digestion of proteins to be analyzed by mass spectrometry - http://www.narrador.embl-heidelberg.de/GroupPages/PageLink/activities/protocols/ingeldigest.html

Category: Proteomic Protocols

In-Gel Digestion Protocol. Excision of protein bands (spots) from polyacrylamide gels Reduction and alkylation . Reduction and alkylation . MALDI analysis of the supernatant after in-gel digestion. Extraction of Peptides.Matthias Wilm EMBL Bioanalytical R - [Read In-gel digestion of proteins to be analyzed by mass spectrometry]

In-Vitro Kinase Assay for Jnk-1/IRS Proteins - http://www.whitelabs.org/Lab%20Protocols/kinase%20and%20phosphatase%20assays/In_vitro%20kinase%20assay%20for%20JNK-1%20IRS%20proteins.htm

Category: Signalling Signal Transduction Protocols: MAPK Protocols

Protocol describing an in vitro assay on the detection of Kinase activity for Jnk-1/IRS proteins. - [Read In-Vitro Kinase Assay for Jnk-1/IRS Proteins]

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.