Identification Of Specific Binding Proteins Research Articles

Improvement of monolithic solid material by utilization of spacer for identification of the target using affinity resins

Affinity chromatography is an important strategy for target identifications. However, commercial available solid materials have limitations while selection of that is sometimes vital for the purpose. We have reported a synthetic resin with a monolithic structure in previous papers. In this paper, introduction effects of spacer to the monolithic material on identification of specific binding protein was quantitatively analyzed using benzensulfonamide as a bait, which exhibited introduction of ω-substituted heptanoic acid as spacer enabled affinity resins to capture the target proteins effectively. Utilization of the optimized spacer enable the monolithic material bearing FK506 to identify not only FKBP12 but FKBP52, calcineurin A and calcineurin B at silver stained level, while that without spacer had failed.

14] Prenylated peptides in identification of specific binding proteins

Publisher Summary This chapter discusses the methods used in the identification of specific binding proteins for prenylated peptides, which was initially identified and characterized using biochemical binding analysis. The chemical cross-linking approaches, which lead to the visualization of membrane proteins that bind prenylated peptides and proteins, are also described. Using prenylated peptides that correspond to C-terminal sequences of proteins containing the CaaX motif as probes, a high-affinity binding protein for the prenylated peptides in the microsomal membrane fraction of bovine brain is identified. The binding protein may serve as the initial component in the processing/trafficking pathway for newly prenylated proteins. The specificity of the binding activity detected in the membrane fraction can be assessed by competition analysis. Typical competitors include prenyl peptides (GG-γ peptide and Farn-ras peptide), unprenylated counterpart peptides, peptides modified by other lipids, and the prenyl diphosphates (GGPP and FPP) at up to 400 n M .

Identification of specific binding proteins for a nuclear location sequence.

The nuclear envelope is a selective barrier against the movement of macromolecules between the nucleus and cytoplasm. Nuclear proteins larger than relative molecular mass 20,000-40,000 are probably actively transported across the envelope through the nuclear pore complex and are directed by specific nuclear location sequences (NLS) in the proteins. NLS mediate the nuclear import of isolated nuclear proteins after microinjection into whole cells and the nuclear accumulation of chimaeric proteins or of non-nuclear proteins conjugated to synthetic peptides. The best-characterized NLS is the simian virus 40 large T-antigen sequence. We have identified two proteins of rat liver by chemical cross-linking that interact with a synthetic peptide containing this sequence: this interaction is specific for a functional NLS, is saturable, and high affinity. The binding proteins are present in a post-mitochondrial supernatant, in nuclei and in a nuclear envelope fraction, which is consistent with a role in the transport of nuclear proteins from the cytoplasm to the nucleus.

5] Identification and assay of platelet-derived growth factor-binding proteins

Publisher Summary This chapter focuses on characteristics of platelet-derived growth factor (PDGF)-binding proteins and factors that affect PDGF-binding protein complexes, identification of specific binding proteins by various assays, and limitations in the analysis of the functional role of these binding proteins. PDGF is a potent mitogen for connective tissue cells, which was discovered because of the ability of platelets to reconstitute the mitogenic activity of whole blood serum lacking in serum derived from cell-free plasma. Release of PDGF and other mitogens from platelet α-granules is dependent on platelet activation, and it has been postulated that focal release of PDGF at sites of injury may play a physiological role in wound healing and tissue repair and a pathological role in the formation of lesions of atherosclerosis characterized by smooth muscle cell proliferation. The possible importance of focal release of PDGF and local PDGF concentrations at the site of release is suggested by in vivo clearance studies with purified and 125 I-labeled PDGF.