Development of chemically stable solid phases for the target isolation with reduced nonspecific binding proteins

Bioorganic and Medicinal Chemistry Letters

Volumn 16, Issue 2, 2006, Pages 447-450

  Takahashi, Teruki ^a   Shiyama, Takaaki ^a   Hosoya, Ken ^b   Tanaka, Akito ^a  

^a Reverse Proteomics Research Institute Co Ltd   (Japan)

^b KYOTO INSTITUTE OF TECHNOLOGY   (Japan)

Author keywords

Affinity chromatography; FK506; FKBP12; FKBP52; Nonspecific binding proteins; Poly(methacrylate)

Indexed keywords

AFFIGEL; DRUG BINDING PROTEIN; FK 506 BINDING PROTEIN; POLYMETHACRYLIC ACID DERIVATIVE; TACROLIMUS; TOYOPEARL; UNCLASSIFIED DRUG;

ABSORPTION; ARTICLE; CONTROLLED STUDY; DRUG DESIGN; DRUG PROTEIN BINDING; DRUG SYNTHESIS; HYDROPHOBICITY; NONHUMAN; PROTEIN ISOLATION; RAT;

ABSORPTION; METHACRYLATES; MOLECULAR CONFORMATION; POLYMERS; TACROLIMUS; TACROLIMUS BINDING PROTEIN 1A;

EID: 28044438201     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2005.09.011     Document Type: Article

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11. The preparation of lysate, binding experimental for capturing the binding proteins, and analysis of proteins were carried out in similar ways to that described in Ref. 3.
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13. 2, dioxane, and water (10 mL, 50:39:10:1) at rt for 15 h, and then washed successively with dioxane, NMP, water, MeOH, and ether, to give 5a (125 mg). The amount of amino group of 5a was determined by the Ninhydrin test and was 2.27 μmol/mg.
14. Western blot analysis on FKBP52 was carried out the following way. The proteins were subjected to SDS-PAGE followed by electroblotting onto PVDF membrane using the Invitrogen XCell II™ blot module. After blocking with Blocking one™ (Nacalai Tesque Inc., cat. 03953-95) for 30 min at rt, the membrane was incubated with anti-FKBP52 IgG (Santa Cruz Biotechnology, Inc., cat. sc-1803) for 1 h at rt. Following washing, the membrane was incubated with HRP-conjugated anti-goat antibodies (Santa Cruz Biotechnology, Inc., cat. sc-2033) for 1 h at rt and washed again. The membrane was soaked for 5 min in the detection reagent ECL plus (Amersham Biosciences Corp. cat. RPN2132). The resulting light was detected on Hyperfilm ECL (Amersham Biosciences Corp. cat. RPN1674K).