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Binding of a ligand to a binding site on protein often triggers a change in conformation in the protein and results in altered cellular function. Hence binding site on protein are critical parts of signal transduction pathways. [10] Types of ligands include neurotransmitters, toxins, neuropeptides, and steroid hormones. [11]
In immunology, epitope mapping is the process of experimentally identifying the binding site, or epitope, of an antibody on its target antigen (usually, on a protein). [1] [2] [3] Identification and characterization of antibody binding sites aid in the discovery and development of new therapeutics, vaccines, and diagnostics.
However, the development of DNA microarrays and fast sequencing techniques has led to new, massively parallel methods for in-vivo identification of binding sites, such as ChIP-chip and ChIP-Seq. [12] To quantify the binding affinity [ 13 ] of proteins and other molecules to specific DNA binding sites the biophysical method Microscale ...
Compared to ChIP-chip, ChIP-seq data can be used to locate the binding site within few tens of base pairs of the actual protein binding site. Tag densities at the binding sites are a good indicator of protein–DNA binding affinity, [14] which makes it easier to quantify and compare binding affinities of a protein to different DNA sites. [15]
Recent advances in chromatin immunoprecipitation followed by sequencing have provided powerful ways to identify genome-wide profiling of DNA-binding proteins and histone modifications. [1] [2] The application of ChIP-seq methods has reliably discovered transcription factor binding sites and histone modification sites.
Methods for functionally mapping epitopes often use binding assays such as western blot, dot blot, and/or ELISA to determine antibody binding. [20] Competition methods look to determine if two monoclonal antibodies (mABs) can bind to an antigen at the same time or compete with each other to bind at the same site. [20]
Binding site identification is the first step in structure based design. [ 20 ] [ 44 ] If the structure of the target or a sufficiently similar homolog is determined in the presence of a bound ligand, then the ligand should be observable in the structure in which case location of the binding site is trivial.
The DNA template labeled at the 3' or 5' end, depending on the location of the binding site(s). Labels that can be used are: radioactivity and fluorescence.Radioactivity has been traditionally used to label DNA fragments for footprinting analysis, as the method was originally developed from the Maxam-Gilbert chemical sequencing technique.