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| They were separated by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a polyvinylidene difluoride membrane which was then blocked with phosphate buffered saline containing 0.1% Tween 20 and 5% skimmed milk. The proteins on the membrane were reacted with a primary antibody (1:1000) (Cell signaling) and then with a horseradish peroxidase-conjugated secondary antibody (1:10000) (Santa Cruz), followed by color development with an ECL western detection reagent (Thermo scientific) to compare the expression level of MAPK (ERK, JNK and p38) with that of β-actin.the reaction was terminated with 0.1 M citrate buffer before absorbance at 405 nm was read in an ELISA reader. |
榮譽版主 (文壇精英)
| 用十二烷基硫酸鈉聚丙烯酰胺凝膠電泳分離它們并轉(zhuǎn)移至氟乙烯膜,然后用含0.1%吐溫和5%脫脂奶的磷酸鹽緩沖液阻塞。膜上的蛋白質(zhì)與一級抗體(1:1000) 起化學(xué)反應(yīng)(細胞信號傳導(dǎo)),然后與辣根過氧化物酶共軛二級抗體 (1:10000) (Santa Cruz), 起化學(xué)反應(yīng),再用ECL免疫印跡檢測試劑(也叫ecl敏發(fā)光液)(Thermo scientific)顯色比較絲裂原活化蛋白激酶(ERK, JNK and p38)與β-actin的表達水平。用酶標(biāo)儀在405nm處讀取吸光度前,用0.1M檸檬酸鹽緩沖液終止反應(yīng)。 |

榮譽版主 (文壇精英)
| 用十二烷基硫酸鈉聚丙烯酰胺凝膠電泳分離它們并轉(zhuǎn)移至氟乙烯膜,然后用含0.1%吐溫和5%脫脂奶的磷酸鹽緩沖液進行阻塞。膜上的蛋白質(zhì)與一級抗體(1:1000) 進行化學(xué)反應(yīng)(細胞信號傳導(dǎo)),然后與辣根過氧化物酶共軛二級抗體 (1:10000) (Santa Cruz)進行化學(xué)反應(yīng),再用ECL免疫印跡檢測試劑(也叫ecl敏發(fā)光液)(Thermo scientific)顯色比較絲裂原活化蛋白激酶(ERK, JNK and p38)與β-actin的表達水平。在用酶標(biāo)儀在405nm處讀取吸光度前,以0.1M檸檬酸鹽緩沖液終止反應(yīng)。 |

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