Cytochrome c Oxidase
René Lontie in Copper Proteins and Copper Enzymes, 1984
Most laboratories, starting from common published procedures, finally arrived at their own modifications depending on the needs of further experimentation.26-28 Several methods are described in detail in Methods in Enzymology.29 Besides repeated fractionation with ammonium sulfate in the presence of detergent as the final step for the purification, not much successful use has been made of ion-exchange chromatographic or of electrophoretic procedures. A method for the ion-exchange chromatographic purification of oxidases from Neurospora crassa and other sources in the presence of detergent has been described.30 Affinity chromatography has been successfully carried out in several laboratories. Thus, a yeast cytochrome c-Sepharose 4B column has been used for the purification of the enzyme from a Triton® X-100 extract of the beef heart mitochondrial membrane.31
Antibodies and Antisera
Lars-Inge Larsson in Immunocytochemistry: Theory and Practice, 2020
The covalent addition of antigens to Sepharose® or polyacrylamide beads (method a) requires the “activation” of the beads by cyanogen bromide treatment, glutaraldehyde, or a variety of other techniques. These model methods have been adopted from affinity chromatography techniques, and numerous methods exist for attaching virtually any substance to the beads. Modem variants of the methods often incorporate a spacer arm (usually consisting of carbon chains) to reduce steric hindrance from the bead surface. Apart from uses in affinity chromatography, such antigen-coated beads have proven useful for solid phase absorption techniques (discussed in Section II.C.2). Such beads have been extensively used for immunocytochemical staining.10,12,68,76 The beads can be made to adhere to glass slides coated with gelatin, and such adherent antigen beads can be immunocytochemically stained just like tissue sections.76 This is a quick and convenient way of screening antisera. Moreover, when the beads are stained by immunofluorescence methods, microspectrofluorometric measurements can be carried out on them, and the DASS system represents a useful standard for many antisera and fluorescent conjugates.12
Approaches for Identification and Validation of Antimicrobial Compounds of Plant Origin: A Long Way from the Field to the Market
Mahendra Rai, Chistiane M. Feitosa in Eco-Friendly Biobased Products Used in Microbial Diseases, 2022
Affinity chromatography is extremely efficient in providing a high degree of purity because it explores a specific region of the protein, that is, the specificity of a binding site (Paiva et al. 2010; Nelson and Cox 2014; Procópio et al. 2017b). It is the chromatographic process of choice if the protein of interest forms a reversible complex with a ligand. For example, a protein that contains a glucose binding site can be isolated using a column containing a glucose polymer. An enzyme can be separated from other proteins using a matrix containing substrate or inhibitor and the enzyme inhibitor will bind to a matrix that has the enzyme immobilized (Coelho et al. 2012; Pontual et al. 2014; Ferreira et al. 2019). When the affinity matrix is not commercially available, it can be produced in the laboratory by immobilizing the binder to an insoluble material such as, for example, Sepharose 4B (Silva et al. 2020). It is important to emphasize that the ligand immobilization must be via functional groups that are not involved in the interaction with the protein of interest. Also the smaller the number of regions for nonspecific binding in the matrix, the greater is its selectivity, that is, its efficiency in selecting the protein among others present in the mixture.
Advances with weak affinity chromatography for fragment screening
Published in Expert Opinion on Drug Discovery, 2019
Fotios Tsopelas, Anna Tsantili-Kakoulidou
Affinity chromatography [27], based on the (bio)selective interactions between two molecules, was first introduced in the late 1960s for protein purification. Typically, the biomolecule in solution (e.g. an enzyme) to be isolated is adsorbed on a column with an immobilized ligand(a specific competitive inhibitor in the case of an enzyme) forming a tight binding complex, whereas non interacting substances (impurities) are washed off. By changing the elution conditions, the substance can be released in a highly purified form. This method enables the separation of a biomolecule practically to 100% purity. The use of affinity chromatography for sample pretreatment or target isolation usually employs a carbohydrate support such as agarose or cellulose. These materials can be easily modified for ligand attachment, can be used with a wide range of elution conditions and have low non-specific binding for many biological compounds. However, they possess limited mechanical stability and relatively low efficiency which means that they are more suitable to work for offline methods and for columns that can be operated at low pressures and flow rates [27].
Toxoplasma gondii infection: novel emerging therapeutic targets
Published in Expert Opinion on Therapeutic Targets, 2023
Joachim Müller, Andrew Hemphill
A key step in the heuristic identification of targets of compounds issued from screening is the characterization of drug-binding proteins [48]. If we admit that a given compound uniquely or at least preferentially physically interacts with a given protein, affinity chromatography is the method of choice to identify such target proteins (Figure 2). The effective compound of interest is coupled to a suitable inert matrix. In parallel, an ineffective compound with high structural similarities to the effective compounds is coupled to the same kind of inert support. Columns filled with this material are inserted into a low-pressure liquid chromatography device. Cell-free extracts from organisms of choice are loaded into both columns in parallel. After suitable washing steps, bound proteins are eluted and identified by convenient proteomic tools. The proteomes identified in eluates from effective and ineffective compounds are compared. The differential affinoproteome, i.e. the subset of proteins binding to effective, but not to ineffective compounds, should then contain potential targets.
Surface-modified polymeric nanoparticles for drug delivery to cancer cells
Published in Expert Opinion on Drug Delivery, 2021
Arsalan Ahmed, Shumaila Sarwar, Yong Hu, Muhammad Usman Munir, Muhammad Farrukh Nisar, Fakhera Ikram, Anila Asif, Saeed Ur Rahman, Aqif Anwar Chaudhry, Ihtasham Ur Rehman
Targetability is an important characteristic in surface-modified polymeric nanoparticles. Different targeting ligands have been added on the surfaces of nanoparticles, which selectively recognize specific membrane receptors on target cells. Surface Plasmon resonance (SPR) technique is utilized to study ligand-receptor molecular association [168]. For example, Barbara and coworkers evaluated the activity of folic acid-containing nanoparticles. Folate binding protein was immobilized on the sensor surface of carboxylated dextran-coated gold film by amine coupling. The nanoparticles were allowed to interact with folate binding protein at different flow rates to study the kinetic parameters of interactions [169]. The presence or absence of targeting ligands on the surfaces of nanoparticles is also determined by affinity chromatography [170]. Bicinchoninic acid assay (BCA) and enzyme-linked immunosorbent assay (ELISA) is used if nanoparticles possess antibodies as targeting agents [171]. The Average number of targeting ligands on the surface of nanoparticles can be investigated by nuclear magnetic resonance (NMR). In this process, the integration values of signals related to targeting ligands are compared with those associated with the rest of nanoparticles [172].
Related Knowledge Centers
- Antibody
- Biomolecule
- Enzyme
- Ligand
- Nucleic Acid
- Protein
- Receptor
- Antigen
- Molecular Binding
- Substrate