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지식나눔

지식나눔

sphingosine kinase assay 방법을 부탁드립니다

2007-06-27 org.kosen.entty.User@5cb4122b 허정선(sundog307)
  • 1
sphingosine kinase assay
  • sphingosine kinase
  • sphingosine 1 phosphate
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    2007-07-02
    • 0
    Radioassay가 주로 활용됩니다. Sphingosine Kinase Assay-- Triton X-100 5% (v/v) was used to prepare a stock solution of 2.5 mM sphingosine. 400 µl of conditioned medium was supplemented to achieve a final concentration of 20 µM sphingosine, 500 µM [32P]ATP (10 µCi), 5 mM MgCl2, 15 mM NaF, 0.5 mM 4-deoxypyridoxine, and 40 mM -glycerophosphate and incubated at 37 °C for 30 min. Lipids were extracted, and samples were resuspended in 50 µl of chloroform. [32P]S1P was resolved by TLC on Silica Gel G60 using 1-butanol/acetic acid/water (60/20/20 v/v) buffer and quantified with a PhosphorImager. Bands corresponding to S1P were also scraped from the plates and counted in a scintillation counter. The intracellular SK activity was obtained using the same assay with 100 µl of crude cell extract (10 µg of protein) complemented with 300 µl of M199 (HUVEC) or Dulbecco's modified Eagle's medium (HEK293).
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    Radioassay가 주로 활용됩니다. Sphingosine Kinase Assay-- Triton X-100 5% (v/v) was used to prepare a stock solution of 2.5 mM sphingosine. 400 µl of conditioned medium was supplemented to achieve a final concentration of 20 µM sphingosine, 500 µM [32P]ATP (10 µCi), 5 mM MgCl2, 15 mM NaF, 0.5 mM 4-deoxypyridoxine, and 40 mM -glycerophosphate and incubated at 37 °C for 30 min. Lipids were extracted, and samples were resuspended in 50 µl of chloroform. [32P]S1P was resolved by TLC on Silica Gel G60 using 1-butanol/acetic acid/water (60/20/20 v/v) buffer and quantified with a PhosphorImager. Bands corresponding to S1P were also scraped from the plates and counted in a scintillation counter. The intracellular SK activity was obtained using the same assay with 100 µl of crude cell extract (10 µg of protein) complemented with 300 µl of M199 (HUVEC) or Dulbecco's modified Eagle's medium (HEK293).
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