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Volumn 21, Issue 2, 2011, Pages 727-729

Chemical affinity matrix-based identification of prohibitin as a binding protein to anti-resorptive sulfonyl amidine compounds

Author keywords

Chemical affinity matrix; Chemical proteomics; Prohibitin

Indexed keywords

5 CHLORO 1 (2,6 DIMETHYLPIPERIDIN 1 YL) N TOSYLPENTAN 1 IMINE; AMIDINE; PROHIBITIN; UNCLASSIFIED DRUG;

EID: 78651256728     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2010.11.123     Document Type: Article
Times cited : (39)

References (27)
  • 15
    • 78651260523 scopus 로고    scopus 로고
    • note
    • 2, 1 mM PMSF, 1% Nonidet P-40 and protease inhibitor cocktail).
  • 18
    • 78651228489 scopus 로고    scopus 로고
    • note
    • Protein sample preparation and its incubation with 2b. RAW264.7 cells were homogenized (1:5, w/v) in buffer A under sonication. The homogenate was then centrifuged at 10,000g for 15 min and 0.5 ml of the supernatant diluted with buffer A (7 mg/ml) was stirred with buffer A-saturated 2b at 4 °C for 15 h. After incubation, the resins were precipitated by centrifugation at 10,000g for 1 min and washed three times with 1 ml of buffer A. The washed beads were then resuspended with 20 μl of SDS sample buffer (100 mM Tris-HCl, 2% sodium dodecyl sulfate, 1% 2-mercaptoethanol, 2% glycerol, 0.01% bromophenol blue, pH 7.6), incubated at 25 °C for 10 min, and subjected to SDS-PAGE followed by the gel staining.
  • 19
    • 78651231787 scopus 로고    scopus 로고
    • note
    • Western blot analysis. RAW264.7 cell lysate (40 μg) were incubated with non-conjugated Affi-Gel 10 or 2b at 4 °C for 15 h and centrifuged at 10,000g for 1 min. The supernatant (unbound fractions) was denatured with SDS sample buffer. Then, the pellets were washed twice with buffer A, resuspended with 20 μl of SDS sample buffer and incubated at 25 °C for 10 min for getting bound fractions. Denaturation of unbound and bound fractions was done by boiling and then samples were subjected to 10% polyacrylamide gels. Electrophoresis was performed using the Mini Protean 3 Cell (Bio-Rad). The resolved proteins were transferred to PVDF membrane (Millipore). The membranes were incubated in blocking buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Tween 20, 3% nonfat dry milk) and then incubated with diluted primary antibodies (1:1000) for 2 h at room temperature. Antibodies used in this study were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz). Following the primary antibody reactions, the membranes were washed with blocking buffer three times (15 min each) and then probed with diluted secondary antibodies (1:2000) for 1 h. The membranes were washed three times (15 min each) and developed with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce Biotechnology) using the LAS-3000 luminescent image analyzer (Fuji Photo Film Co., Ltd).


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.