title: A quantitative analysis and chemical approach for the reduction of nonspecific binding proteins on affinity resins.
abstract: Tubulin and actin often bind nonspecifically to affinity chromatography resins, complicating research toward identifying the cellular targets of small molecules. Reduction of nonspecific binding proteins is important for the success of such biochemical approaches. To develop strategies to circumvent this problem, we quantitatively investigated the binding of tubulin and actin to a series of affinity resins bearing 15 variant ligands on 3 commercially available polymer supports. Nonspecific protein binding was proportional to the hydrophobicity of the affinity resins and could be quantitatively correlated to the CLOGP values of the ligands, which are a measure of compound hydrophobicity. When compounds had CLOGP values greater than 1.5, (amount of tubulin) = 0.73 x CLOGP - 1.1 (n = 7, r = 0.97), and (amount of actin) = 0.42 x CLOGP - 0.79 (n = 7, r = 0.99). On the basis of these studies, we designed a novel hydrophilic poly(ethylene glycol) (PEG) spacer (26) for the conjugation of ligands to chromatography resins. As predicted by our binding algorithm, introduction of this spacer reduced the amount of nonspecific protein binding in proportion to the number of ethylene glycol units.
authors: Chemistry Department, Reverse Proteomics Research Institute Co., Ltd. 2-6-7 Kazusa-Kamatari, Chiba 292-0818, Japan.
authors: Tsuruki
authors: T
authors: Tamura
authors: Tomohiro
authors: T
authors: Terada
authors: Akito
authors: A
authors: Tanaka
mesh_terms: D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D008024:Ligands; D015394:Molecular Structure; D011108:Polymers; D011485:Protein Binding; D012117:Resins, Synthetic / Q000737:chemistry / Q000378:metabolism*; D016559:Tacrolimus / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
chemical_list: D000199:Actins; D008024:Ligands; D011108:Polymers; D012117:Resins, Synthetic; D014404:Tubulin; D016559:Tacrolimus