binding Protocols

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

The ABH crosslinker binds to the Fc portion of an antibody molecule, away from the antigen binding site, resulting in a divalent immunologically active immunoglobulin derivative. Pierce - [Read ABH (p-Azidobenzoyl hydrazide) PDF]

Category: Cytoskeleton Protocols: Actin Myosin Protocols

Protocol includes: Binding assays for tropomyosin, villin or caldesmon and Bundling Assays - [Read Actin Binding & Bundling Sedimentation Assays]

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: 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: PCR Protocols: Reverse Transcriptase RT-PCR Protocols

Protocol uses a single thermostable RNA polymerase to perform high-specificity RT-PCR.  A high-temperature RT reaction is followed by PCR amplification of the cDNA using a single thermostable poymerase, the GeneAmp AccRT RNA PCR enzyme from Applied Biosystems.  The high temperature of the RT reaction enhances the specificity of primer binding and also reduces secondary structure in the template, thereby increasing the efficiency of polymerization. - [Read Amplification of RNA: High-Temperature Reverse Transcription and DNA Amplification with a Magnesium]

Category: Molecular Biology Protocols: Carbohydrate Protocols

High performance liquid affinity chromatography (HPLAC) is a useful procedure to investigate he interactions between carbohydrate binding protein and their ligands. Technical requirements are similar to conventional HPLC. HPLAC can screen and separate natural ligands from complex biological mixtures. WeiTong Wang~GlycoTech Corporation, Rockville, Maryland - [Read Analysis of Oligosaccharide Ligands by High Performance Liquid Affinity Chromatography]

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: 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: Immunology: Chemotaxis

AS Ammonium Sulfate Binding Assay for Cells. Devreotes Lab, John Hopkins Medical Centre. - [Read AS Ammonium Sulfate Binding Assay for Cells]

Category: Antibody Protocols: Antibody Labeling Protocols

Once tissues are fixed and permeabilized, the antibodies are added. These antibodies can be labeled directly or detected by a labeled secondary reagent. For indirect detection, any reagent that binds specifically to the primary antibody can be "tagged" and used to locate the antibody. The possible reagents include anti-immunoglobulin antibodies, protein A or G, or, if the first antibody is labeled with biotin, streptavidin. They can be labeled with enzymes or gold. - [Read Binding Antibodies to Tissue Sections Protocol]

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

The bradford dye-binding assay is a colorimetric assay for measuring total protein concentration.  It involves the binding of Coomassie Brilliant blue to protein. There is no interference from cations nor from carbohydrates such as sucrose. 
Detergents such as sodium dodecyl sulfate and triton x-100 can interfere with the assay, as well as strongly alkaline solutions.

Includes a general overview of the procedure and preparation of the standards in the protocol. - [Read Bradford Assay Method]

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: Mouse Protocols

Protocol for calcium dependent by pentraxins to ligands immobilized on agarose, and effect of C-reactive protein on leptin action in mice in vivo. Includes: Testing for binding of CRP to immobilized leptin; Testing for binding of radiolabelled leptin by immobilized CRP, SAP and leptin receptor; Testing for effect of human CRP on human leptin action in mice. - [Read Calcium Dependent by Pentraxins to Ligands, and Effect of C-reactive Protein on Leptin Action]

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: Cell Biology and Cell Culture: Apoptosis Protocols: Apoptosis Analysis

Detection of Phosphatidylserine Externalization During Apoptosis. Shailaja Kasibhatla et al. An early event in apoptosis is the externalization of phosphatidylserine (PS), a phospholipid normally restricted to the inner leaflet of the plasma membrane. This apoptotic event can be monitored using Annexin V, a PS-specific binding protein. This protocol uses Annexin V-FITC as a probe, but Annexin V-biotin is also available, and binding can be revealed using streptavidin-FITC or oth - [Read Detection Of Phosphatidylserine Externalization During Apoptosis (Subscription Required)]

Category: Chromatography Protocols: DNA RNA Affinity Chromatography

DNA Affinity Chromatography, DNA affinity chromatography can be a low-tech method using gravity flow at 4°C, a disposable chromatography column, and DNA affinity resin prepared in the laboratory (see Preparation of a DNA Affinity Column). Include 10-20% glycerol and 0.025-0.1% NP-40 in the column buffers to suppress losses due to nonspecific adsorption of protein to surfaces. Load the protein in a buffer that is compatible with binding of the protein to its target site. Keith Brocklehurst et al - [Read DNA Affinity Chromatography Using Gravity Flow - Subscription Required]

Category: Nucleic Acid Purification Protocols: DNA Isolation Protocols: Genomic DNA Isolation Protocols

In this protocol, the DNA-binding capacity of Wizard MagneSil particles is used to capture and release a consistent amount of DNA (100 ng) across a wide range of samples.  At the end of the procedure, the DNA is eluted into 100 µl Elution Buffer to give a final concentration of 1 ng/µl, relieving the need for postpurification DNA quantitation. - [Read DNA IQ Isolation of Genomic DNA from Stains and Buccal Swabs Protocol]

Category: DNA Protocols: EMSA DNA-Protein Protocols

Magnetic DNA affinity purification of yeast transcription factor tau--a new purification principle for the ultrarapid isolation of near homogeneous factor. Gabrielsen et al. 1989 - [Read DNA-Binding Protein Purification with Dynabeads.]

Category: DNA Protocols: EMSA DNA-Protein Protocols

DNaseI Footprinting Assay. Works well high affinity dna binding proteins. Breeden Lab. - [Read DNaseI Footprinting Assay]

Category: RNA Protocols: RNA-binding Protein Purification

Dynal Bead RNA-Binding Protein Purification. DYNAL. - [Read Dynal Bead RNA-Binding Protein Purification]

Category: DNA Protocols: EMSA DNA-Protein Protocols

Primer design, Primer annealing, Primer labelling, Probe purification, In vitro hybridization DNA-protein Binding reaction. Jonathan Flint. - [Read EMSA Protocol using 32-p.]

Category: Developmental Biology: Embryology Protocols

The FAM caspase binding assay kits from ATCC Corporation can be used to determine amounts of active caspases in cells. The FAM-labeled caspase inhibitor can freely diffuse into the cell. Active caspase irreversibly binds the inhibitor. Upon washing the cells, the amount of fluorescence is proportional to the amount of active caspase in the cell. FAM-LETD-fmk (catalog no. 30-1306) is used to detect caspase 8 and FAM-LEHD-fmk (catalog no. 30-1308) is used for caspase 9. - [Read Fam Caspase 8 and 9 Binding Assay for Embryos Protocol]

Category: Cell Biology and Cell Culture: Flow Cytometry Protocols

FIXATION and DNA Staining for Cell Cycle Analysis Protocol. This method of DNA staining utilizes ethanol to fix the cells and permeabilize the membrane, which allows the dye (Propidium Iodide) to enter the cells.  Propidium Iodide (PI) is a DNA-binding fluorochrome that intercalates in the double-helix.  Ribonuclease-A is used to eliminate the staining of double-stranded RNA. - [Read FIXATION and DNA Staining for Cell Cycle Analysis]