Mechanisms of Tumor Cell Invasion studied In Vitro
Rolf Bjerkvig in Spheroid Culture in Cancer Research, 2017
For those enzymes to which antibodies have been raised, immunological methods of protein identification (immunoprecipitation, western blots) are of utility. Stephens et al.79 have applied immunodetection in a microwell and substrate format to develop an immunocapture method. In this method, antibodies are used to capture the enzyme, and quantitation is achieved by the subsequent addition of substrate, incubation, and the colorimetric assessment of remaining nondegraded substrate. This method has been applied to the detailed study of the cellular location of plasmin degradation at the cell surface of sarcoma cells by plasminogen activator bound to the cell membrane.80 The results from this investigation indicate that membrane-bound enzymes serve directly and indirectly, by way of cascade initiation, in determining the proteolytic environment of tumor cells.
Experimental Strategies
Clive R. Bagshaw in Biomolecular Kinetics, 2017
Finding the natural ligands and functions of such expressed macromolecules is not trivial, even when three-dimensional atomic structures are available. Functional properties are often proposed on the basis of their similarity in structure to known proteins. At a minimum, this approach suggests potential ligands/substrates that might be tested for interaction. Proteins almost invariably function via a binding interaction with a small ligand or another macromolecule, and therefore, methods to study binding are at the heart of many assays. Interacting partners may be identified by co-immunoprecipitation from crude cell lysates [320]. Some practitioners describe this method as the gold standard in identifying interactions, but it is limited to high-affinity binding with a slow dissociation rate that can withstand the washing steps. Co-immunoprecipitation is therefore used primarily as a qualitative screening assay. Quantitative equilibrium binding methods to determine Kd values are usually the next step in the characterization [321], although in the case of high-affinity interactions, kinetic assays may be easier to perform [322]. In any event, it is useful to know the order of magnitude of the equilibrium binding constant in order to choose the most appropriate kinetic assays for further analysis. We therefore start with a review of equilibrium methods, many of which can readily be extended to yield kinetic information.
Immunological Tests for Diagnosis of Disease and Identification of Molecules
Julius P. Kreier in Infection, Resistance, and Immunity, 2022
Some clinically significant proteins occur at concentrations too low to be detected by immunoprecipitation methods. In 1960 Yalow and Berson introduced radioimmunoassay (RIA) which is a sensitive and specific technique. This method permits measurement of very low concentrations of any material to which an antibody can be raised. Sensitivity results from the low levels of radioactivity which can be measured. The specificity of the assay is determined by the specificity of the antibody to which the radioisotope is coupled. A more specific antibody is usually required for RIA than for precipitation assays.
Involvement of β-catenin in Androgen-induced Mesenchymal Transition of Breast MDA-MB-453 Cancer Cells
Published in Endocrine Research, 2021
Mamoun Ahram, Randa Bawadi, Mohammad S. Abdullah, Dana B. Alsafadi, Haneen Abaza, Sallam Abdallah, Ebtihal Mustafa
Pierce Co-Immunoprecipitation kit (Thermo Fisher) was used for co-immunoprecipitation of β-catenin and AR. The antibody immobilization and protein co-immunoprecipitation were performed according to the manufacturer’s instructions. Anti-β-catenin antibody was immobilized and immunoblotting was performed using anti-AR and -β-catenin antibodies. A goat anti-mouse antibody was immobilized on the resin (ab97023; Abcam, Cambridge, UK). Briefly, after 24 hrs of seeding, cells were treated with DHT for 1, 4, 16, or 72 hrs. Cells were scraped in ice-cold lysis/wash buffer and the lysates were centrifuged at 13,000 g for 10 min and transferred into clean micro-centrifuge tubes. The protein lysates were mixed with the appropriate resin and incubated with gentle mixing overnight at 4°C. The spin columns were placed into collection tubes, elution buffer was added to the columns and incubated for 5 min at room temperature, and then centrifuged to elute the antibody-bound proteins. The elutes were stored at −80°C until used for immunoblotting analysis.
Proteogenomic interrogation of cancer cell lines: an overview of the field
Published in Expert Review of Proteomics, 2021
Olson Tsang, Jason W. H. Wong
The CCL protein interactome, which includes protein–protein, protein–RNA, and protein–DNA interactions, has also been investigated. The traditional approach is to use immunoprecipitation to enrich protein complexes using an antibody for the target protein [46]. Alternatively, proteins that interact with specific nucleic acid sequences have been identified with DNA or RNA probes complementary to those sequences [47]. In recent years, proximity-based labeling using labeling enzymes such as biotin ligase (BioID) or ascorbate peroxidase (APEX) conjugated to a protein or RNA of interest has been widely used to study protein-protein [48] and protein-RNA [49] interactions, respectively. The popularization of dead CRISPR associated proteins (dCas) has further enabled the study of the proteome interacting with specific DNA and RNA sequences using dCas9 [50] and dCas13 [51], respectively.
The Antibody Society’s antibody validation webinar series
Published in mAbs, 2020
Jan L.A. Voskuil, Anita Bandrowski, C. Glenn Begley, Andrew R.M. Bradbury, Andrew D. Chalmers, Aldrin V. Gomes, Travis Hardcastle, Fridtjof Lund-Johansen, Andreas Plückthun, Giovanna Roncador, Alejandra Solache, Michael J. Taussig, James S. Trimmer, Cecilia Williams, Simon L. Goodman
Antibodies targeting native-folded proteins may well show different selectivity when used for detection of unfolded, or partially unfolded proteins. Unfolding may unmask the epitope in another protein, or hide it in the designated target. Similarly, antibodies against denatured proteins may show different selectivity when used for detection of folded proteins. The level of unfolding of proteins differs in WB, IHC, ICC and in antigen-coated microwells for enzyme-linked immunosorbent assay (ELISA). The level of natively folded protein may differ in samples used in immunoprecipitation (IP), in capture ELISA, and in multiplex systems. In microwells for ELISA, the level of correct folding can depend on the size and chemical characteristics of the protein, the plastic surface of the well, and the pH and ionic conditions at which adsorption and assay are performed. It is worth noting that a higher level of selectivity can be enforced when antibodies are used in a dual-recognition combination, as in sandwich assays (two antibodies per protein), which can enhance the reliable detection of a target antigen. In such cases it may be acceptable to use a less specific (polyclonal) antibody i.e., for capture, combined with a highly specific (monoclonal) antibody i.e., for detection.