proteins Protocols

Category: Primary Cell Isolation and Culture Protocols: Laser Capture Microdissection Protocols: Tissue Preparation for LCM Protocols

LCM isolates specific cells or tissues from samples mounted on microscope slides. The samples are viewed through a thermoplastic film that is attached to a microcentrifuge tube lid. Localized heat, caused by the application of a laser pulse, fuses the membrane to the cells of interest, which can then be harvested for further analysis. RNA and proteins can be purified from the isolated cells, allowing detailed analysis of gene expression. This protocol is divided into three stages. - [Read (LCM): Preparation and Sectioning of Frozen Tissue Blocks and Purification of RNA from Isolated Cel]

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

Protocol for 2-D polyacrylamide gel electrophoresis. Includes: 50 ml IEF Lysis Buffer; 10X Electrophoresis Running Buffer (10 L); 30% Acrylamide Stock (1 liter); Separating Acrylamide Gel; 50 ml Equilibration Buffer I; 50 ml Equilibration Buffer II; Transfer Buffer; Sample Preparation; Tissue Processing; Reswelling; Prepare Gradient Acrylamide Gel (9-18%); Transfer and Sequencing of Proteins. - [Read 2-D Polyacrylamide Gel Electrophoresis Protocol]

Category: Bioinformatics

Using AFPredictor, it was demonstrated that ‘ordered surface carbons’ (OSCs) are a distinguishing feature of AFPs and, more specifically, their ice-binding surfaces. AFPredictor identified AFPs from within a large set of structures with greater than 99% specificity. Furthermore, it was used to identify a novel ice-binding protein by screening a library of homology modeled structures based on cDNA sequences obtained from cold-acclimated winter rye (Secale cereale). - [Read A Computational Screening protocol for Antifreeze/Ice-Structuring Proteins]

Category: RNA Protocols: RNA-binding Protein Purification

A high yield affinity purification method for specific RNA-binding proteins: isolation of the iron regulatory factor from human placenta. Neupert 1990. - [Read A high yield affinity purification method for specific RNA-binding proteins.]

Category: Molecular Biology Protocols: Carbohydrate Protocols

Solvent partition protocol allows the isolation of gangliosides from small samples and from samples where ganglioside concentrations are low, especially relative to the concentration of potentially contaminating proteins and other large molecular weight species. Stephan Ladisch Director, Center for Cancer and Transplant Biology, Children's National Medical Center, Washington, D.C. - [Read A Method for Micro-Scale Isolation and Purification of Gangliosides]

Category: Protein Protocols: Protein Quantitation Concentration Protocols

Absorbance assays are fast and convenient, since no additional reagents or incubations are required.  No protein standard need be prepared.  The assay does not consume the protein.  The relationship of absorbance to protein concentration is linear.  Because different proteins and nucleic acids have widely varying absorption characteristics there may be considerable error, especially for unknowns or protein mixtures. - [Read Absorbance Assay 280 nm]

Category: Signalling Signal Transduction Protocols: MAPK Protocols

MAPK Protocols. Resolving Phosphorylated MAPK Proteins by SDS–PAGE, Filter Binding Assay for activated MAPK γ-32P incorporation of MAPK. Stratagene - [Read Activated MAP Kinase Protocols Stratagene PDF]

Category: Viral Culture and Isolation Protocols: Viral Isolation Protocols

Protocol describes large fragment DNA replication by Adenovirus proteins using TP-DNA as template. - [Read Adenovirus DNA Replication Assay Protocol]

Category: RNA Protocols: RNA-binding Protein Purification

Affinity Chromotography Magnetic Bead Purification of RNA-binding proteins from Rat Brain. Scaturro et al. 2003. - [Read Affinity Chromotography Magnetic Bead Purification of RNA-binding proteins from Rat Brain.]

Category: Protein Protocols: Protein Electrophoresis

An excellent guide on the analysis of proteins on SDS-PAGE gels, through staining with coomassie blue dye and western blot analysis. Analysis of Protein Gels (SDS-PAGE). David R. Caprette, Rice University. - [Read Analysis of Protein Gels (SDS-PAGE)]

Category: Protein Protocols: Immunoprecipitation Protocols

Antibody-antigen complexes are removed from solution by addition of an insoluble form of an antibody binding protein such as Protein A, Protein G or second antibody. Immunoprecipitation protocols / methodology and technical background information. P.J. Ha - [Read Analysis of Proteins by Immunoprecipitation]

Category: Proteomic Protocols: 2-D Gel Electrophoresis

Analysis of Proteins using Small Format 2D Gel Electrophoresis. Cash Lab - [Read Analysis of Proteins using Small Format 2D Gel Electrophoresis]

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

Annexin V, belonging to a recently discovered family of proteins, the annexins, with anticoagulant properties has proven to be a useful tool in detecting apoptotic cells since it preferentially binds to negatively charged phospholipids like PS in the presence of Ca2+ and shows minimal binding to phosphatidylcholine and sphingomyeline. Changes in PS asymmetry, which is analyzed by measuring Annexin V binding to the cell membrane, were detected before morphological changes associated with... - [Read Annexin V Protocol]

Category: Protein Protocols: Protein Expression Protocols

Baculovirus Production for Recombinant Proteins. Protocol is a modified version of the pharmingen Baculogold kit. Brian Dynlacht. Mike A. Dyer Lab. - [Read Baculovirus Production for Recombinant Proteins]

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

Peroxisomes of higher eukaryotes, glycosomes of kinetoplastids, & glyoxysomes of plants are related microbody organelles that perform differing metabolic functions tailored to their cellular environments. The close evolutionary relationship of these organelles is most clearly evidenced by the conservation of proteins involved in matrix protein import and biogenesis. glycosome can be viewed as an offshoot of the peroxisomal lineage with additional metabolic functions, specifically glycolysi - [Read Biogenesis and Function of Peroxisomes and Glycosomes]

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

Includes Abbreviations, Background, and Procedure steps using BSA. The Bradford protein assay (1) is one of several simple methods commonly used to determine the total protein concentration of a sample.  The method is based on the proportional binding of the dye Coomassie to proteins. The assay is colorimetric; as the protein concentration increases, the color of the test sample becomes darker.  Coomassie absorbs at 595 nm.  - [Read Bradford Protein Concentration Assay]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

Protocol uses specific antibodies coupled to one of four fluorochromes: fluorescein isothiocyanate (FITC), R-phycoerythrin (PE), peridinin chlorophyll-a (PcP), and allophycocyanin (APC). These fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer. - [Read Characterization of Cells by Flow Cytometry Protocol]

Category: Immunology: B Cell Protocols

Fluorochromes can be used simultaneously to stain and analyze the expression patterns of four different proteins in the same sample. The fluorochrome stained cell populations are analyzed using a FACSCalibur dual-laser flow cytometer. - [Read Characterization of Cells by Flow Cytometry Protocol]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Method to identify genomic targets of DNA-binding factors. Chromatin immunoprecipitation (ChIP) assay on high-throughput microarray based methods for discovering genomic regions occupied by human DNA-binding proteins. Oberley and Farnham. - [Read ChIP on ChIP Protocol PDF]

Category: Immunology: Immunoprecipitation Protocols: Chromatin Immunoprecipitation Protocols

Protocol describes the use of chromatin immunoprecipitation technology (ChIP) to analyze interactions of proteins or protein complexes with DNA in vivo. In this approach, the material is fixed with formaldehyde to preserve DNA-protein and protein-protein associations, the cells are lysed, and the chromatin is cut and solubilized. The chromatin suspension is immunoprecipitated with an antibody against the protein(s) of interest, and the coimmunoprecipitated DNA fragments are analyzed. - [Read Chromatin Immunoprecipitation (ChIP) of Protein Complexes Protocol]

Category: Epigenetics and Methylation Protocols: ChIP Chromatin Immunoprecipitation Protocols

Chromatin Immunoprecipitation Protocol for Histone Modification Chromatin and Associated Proteins. Roderick O’Sullivan & Joost Martens. Chromatin Immunoprecipitation (ChIP) experiments are routinely performed in many laboratories around the world to examine the occupancy of proteins or chromatin modifications over particular stretches of the genome. - [Read Chromatin Immunoprecipitation Protocol for Histone Modification Chromatin and Associated Proteins]

Category: DNA Protocols: Chromatin Immunoprecipitation Protocols

Mapping Protein/DNA Interactions by Cross-Linking Examining the Distribution of Telomeric and DNA Repair Proteins by ChrIP and Real-Time PCR - [Read Chromatin-IP (ChrIP) Protocol]

Category: Chromatography Protocols: Column Chromatography Protocols

Size Exclusion Column Chromatography Protocol. PDF. In this protocol you will learn how to use three types of column chromatography: Gel Filtration or Size Exclusion (SEC), Ion Exchange (IEC), and affinity (AC) in order to purify proteins and enzymes based on the physical properties of these biomolecules. Univ. Arizona, Biochemistry. - [Read Column Chromatography Protocol]

Category: Cytogenetics Protocols: In Situ Hybridization Protocols

Protocol for combined DNA in situ hybridization and immunocytochemistry for the simultaneous detection of nucleic acid sequences, proteins, and incorporated BrdU in cell preparations. Includes: Cell preparations and BrdU labeling; Detection of antigen by immunocytochemistry (ICC); Visualization of ICC antigen; -Gal-BCIG reaction (for producing a blue precipitate visible under brightfield microscopy); Cell processing for in situ hybridization; In situ hybridization (ISH); etc... - [Read Combined DNA In Situ Hybridization and Immunocytochemistry Protocol]

Category: Reporter Gene Assays: GFP Assay Protocols

The following protocol can be used for the development of stable cell lines expressing GFP fusion proteins. Although optimal transfection procedures (e.g., calcium phosphate, electroporation, or FuGENE 6 [Roche Applied Science]) vary depending on cell type, this general transfection procedure has been successful for stable transfection of HeLa, A-431, U2OS, BHK, and HT1080 cells. - [Read Constructing and Expressing GFP Fusion Proteins]