Affinity Modification
Affinity Modification - Related Videos
12:11
Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization
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Cited by 9 •
2020
This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.
03:07
Electron Affinity
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2020
The electron affinity (EA) is the energy change for adding an electron to a gaseous atom to form an anion (negative ion). This process can be either endothermic or exothermic, depending on the element. Many of these elements have negative values of EA, which means that energy is released when the gaseous atom accepts an electron. However, for some elements, energy is required for the atom to become negatively charged, and the value of their EA is positive. Just as with ionization energy,...
01:41
Affinity and Avidity
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2019
Overview Antibodies bind to toxins or substances on the surface of cells, bacteria, viruses, or fungi. The substance is called an antigen, and the precise binding site is the epitope. The strength of the antibody-epitope interaction is called affinity. When an antibody binds an antigen by multiple epitopes, the cumulative strength of the interaction is called avidity. The strength of the interaction influences the elicited immune response. The Adaptive Immune System Increases Efficiency by...
02:32
Histone Modification
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2020
The histone proteins have a flexible N-terminal tail extending out from the nucleosome. These histone tails are often subjected to post-translational modifications such as acetylation, methylation, phosphorylation, and ubiquitination. Particular combinations of these modifications form “histone codes” that influence the chromatin folding and tissue-specific gene expression. Acetylation The enzyme histone acetyltransferase adds acetyl group to the histones. Another enzyme, histone deacetylase,...
10:07
Identification of Post-translational Modifications of Plant Protein Complexes
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Cited by 8 •
2014
We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.
FAQs
The crucial sequence is recognition followed by reaction. Molecular complementarity positions the analog in a ligand-binding pocket, bringing its reactive group close to an amino acid residue. That proximity enables covalent attachment at a selected location rather than relying only on reversible association. The chemical label therefore reports both binding-site geometry and the local reactivity of the biomolecule.
Specificity depends on whether the analog is accepted by the same structural features that recognize the natural substrate, ligand, or cofactor. A well-matched analog can place its reactive group near the intended residue, whereas poorly matched chemistry may reveal nonspecific reactivity. This distinction matters because a covalent label is informative only when binding-guided positioning, not chemical reactivity alone, determines attachment.
Because the bond persists after the initial recognition event, the modified molecule retains a stable mark at the contacted site. Investigators can use that mark to connect a particular residue or region with ligand binding and functional behavior. This is especially valuable for separating residues that shape an active site from residues that merely react chemically without contributing to recognition.
A practical workflow begins by selecting a reactive analog related to the substrate, ligand, or cofactor of interest. The analog is then allowed to interact with the target biomolecule so molecular complementarity can position it near a residue. Covalent attachment produces a modified molecule whose structural and functional consequences can be examined to map binding-site features.
Mapping residues near a binding site gives bioengineers a structural basis for redesigning an enzyme. Changes can be planned around features associated with substrate or cofactor recognition and evaluated for effects on catalytic activity or selectivity. The method therefore links chemical evidence about binding-site architecture to protein-engineering goals rather than treating sequence changes as unguided modifications.
For an enzyme or receptor used in a biosensor, identifying ligand-contacting regions can help determine which molecular features control recognition. That information can support selection or redesign of proteins with more appropriate binding behavior, while distinguishing specific from nonspecific labeling helps prevent incidental chemical reactivity from being interpreted as evidence of sensing function.