SPA bead selection kit for receptor binding studies (contains 100 mg of WGA PVT, WGA Ysi, WGA PEI type A, WGA PEI type B, and Poly-L-lysine beads)
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The treatment of PVT-WGA SPA beads with positively charged polyethyleneimine (PEI) blocks potential non-specific binding sites on the SPA bead surface. There are two SPA bead types available with PEI treatment. The PVT-WGA-PEI type A SPA beads are treated with PEI prior to the coupling of WGA to the PVT SPA bead. The PVT-WGA PEI type B SPA beads are treated with PEI after the WGA coupling stage. The PVT-WGA-PEI type A and type B SPA beads exhibit different characteristics with regard to the non-specific binding of radiolabelled ligand directly to the SPA bead. Therefore, both bead types should be evaluated when deciding which SPA bead to use and both are included in the Select-a-Bead Kit. The binding capacity of both bead types for cell membrane protein remains 10–30 μg membrane protein per milligram of SPA bead.
SPA Scintillation beads are microspheres containing scintillant which emit light in the blue region of the visible spectrum. As a result, these beads are ideally suited to use with photomultiplier tube (PMT) counters such as the MicroBeta2 or TopCount.
Two types of core SPA Scintillation bead are available - yttrium silicate (YSi) and Polyvinyltoluene (PVT). PVT beads are plastic, larger in size, and stay in suspension longer than the crystalline YSi beads.
Scintillation proximity assay (SPA) is a homogeneous and versatile technology for the rapid and sensitive assay of a wide range of biological processes, including applications using enzyme and receptor targets, radioimmunoassays, and molecular interactions. When 3H, 14C, 33P, and 125I radioisotopes decay, they release β-particles (or Auger electrons, in the case of 125I). The distance these particles travel through an aqueous solution is dependent on the energy of the particle. If a radioactive molecule is held in close enough proximity to a SPA Scintillation Bead or a SPA Imaging Bead, the decay particles stimulate the scintillant within the bead to emit light, which is then detected in a PMT-based scintillation counter or on a CCD-based imager, respectively. However, if the radioactive molecule does not associate with the SPA bead, the decay particles will not have sufficient energy to reach the bead and no light will be emitted. This discrimination of binding by proximity means that no physical separation of bound and free radiochemical is required.
|Bead Type or Core Bead Type||PVT (Scintillation),YSi (Scintillation)|
|Coating Treatment||WGA, Poly-lysine, PEI|
|Quantity in a Package Amount||5.0 Units|
|Unit Size||100 mg|
Interactions between proteins are a key feature of many biochemical processes, for example cell signalling. In the absence of any enzymatic activity, measurement of protein:protein interactions has presented problems. Scintillation Proximity Assay (SPA) technology permits the direct measurement of binding of one protein to another.
Many proteins are able to recognize and subsequently bind DNA. These proteins were first identified by their presence in isolated DNA complexes, by their ability to bind DNA in vitro, and by their absolute requirement in many DNA-dependent functions.
SH2 and SH3 domains are small, independent domains of about 100 or 70 amino acid residues respectively. They are foundin a variety of proteins, and can occur together or separately. SH2 domains are thought to be involved in signal transduction mechanisms. Some SH2 domains control enzyme activity by binding membrane-bound receptors to regulate downstream events such as kinase cascades.
The Cytostar-T plate enables the study of cell cultures which normally grow as an adherent monolayer on culture treated plastic or on a layer of extracellular matrix proteins. This system is therefore potentially suitable for carrying out ligand binding assays in an homogeneous format without disturbing the equilibrium between bound and free ligand, and whilst maintaining the cells in a more physiological environment.