Design and synthesis of a biotin-tagged photoaffinity probe of paeoniflorin

Bioorganic and Medicinal Chemistry Letters

Volumn 16, Issue 12, 2006, Pages 3306-3309

  Qiu, Wen Wei ^a   Xu, Jie ^a   Liu, Da Zhi ^a   Li, Jing Ya ^a   Ye, Yang ^a   Zhu, Xing Zu ^a   Li, Jia ^a   Nan, Fa Jun ^a  

^a SHANGHAI INSTITUTE OF MATERIA MEDICA   (China)

Author keywords

Paeoniflorin; Photoaffinity probe; Synthesis

Indexed keywords

BIOTIN; PAEONIFLORIN;

ANIMAL TISSUE; ARTICLE; BINDING AFFINITY; DRUG DESIGN; DRUG SYNTHESIS; LABELING INDEX; NONHUMAN; PHOTOAFFINITY LABELING; PROTEIN TARGETING; PURIFICATION; RAT; STRUCTURE ACTIVITY RELATION;

ANIMALS; BENZOIC ACIDS; BIOTIN; BRIDGED COMPOUNDS; DRUG DESIGN; GLUCOSIDES; MOLECULAR STRUCTURE; PHOTOAFFINITY LABELS; RATS;

ANIMALIA;

EID: 33646166121     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2006.03.021     Document Type: Article

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23. Homogenate preparation of rat cortex. Rats were sacrificed by cervical dislocation and brains were removed and immediately placed in ice-cold saline. After dissection of the cortex, tissues were homogenized in 15 volumes of 0.32 mol/L sucrose using a glass/Teflon homogenizer. The homogenate was centrifuged at 1000g for 10 min, and the supernatant was centrifuged at 36,000g for 30 min and then centrifuged at 48,000g for 10 min. The precipitate was resuspended in 30 volumes of 50 mM Tris·HCl buffer (pH 7.4), and centrifuged at 48,000g for 10 min. Finally, precipitate was resuspended in 5 volumes of 50 mM Tris·HCl buffer (pH 7.4) and stored at -80 °C. The protein concentration of the suspension was measured according to Bradford method.
24. Photoaffinity labeling and streptavidin detection. The cortex homogenate was diluted to 2.5 mg/mL with 50 mM Tris·HCl buffer (pH 7.4). The labeling reaction was initiated by incubating homogenate with the probe (dissolved in DMSO) at 4 °C for 8 h, and then exposed to UV 365 nm (220v, 6 W, 365 nm) at a distance of 3 cm. The reaction mixture was centrifuged at 48,000g for 10 min, then the supernatant was removed and the precipitate was resuspended in lysis buffer (urea 480 mg/L, chaps 40 mg/L, Tris-base 4.8 mg/L, DTT 10 mg/L, Ampholate 50 ml/L, and bromophenol blue 0.002%) at 4 °C for 1 h and centrifuged at 18,000g for 2 h. The supernatant was dialyzed against 50 mM Tris·HCl buffer (pH 7.4). To detect the proteins photo-cross-linked by probe, the homogenate photolabeled with probe was subjected to SDS-PAGE electrophoresis and then transferred onto polyvinylidene fluoride membrane. The membrane was blocked with 1% BSA in PBS with 0.05% Tween 20 at room temperature for 1 h, washed using PBS with 0.05% Tween 20 for 5 min by two times, and then incubated with streptavidin-HRP polymer conjugate (Sigma, Cat.S 2438) diluted 1:10,000 in PBS with 0.05% Tween 20 at room temperature for 1 h. After six washes for 5 min each using PBS with 0.05% Tween 20, the membrane was treated with Enhanced ChemiLuminescence (ECL, Amersham) detection reagents. Biotinylated proteins were visualized by exposure to X-ray and imaging development.