Solutions CPRG Assay Buffer 50 mM KPO4 pH 7.5 5 ml 1M KPO4 pH 7.5 1 mM MgCl2 0.1 ml 1M MgCl2 up to 100 ml with Q store at room temperature 1M KPO4 pH 7.5 250 ml 1M KH2PO4 (monobasic) 68.05 g up to 500 ml 100 ml 1M K2HPO4 (dibasic) 87.1 g up to 500 ml 50 mM CPRG 0.0304 g chlorophenyl red b-galactopyranoside (Boehringer Mannheim #834-308) up to 1 ml with Q store in the dark at -20° Procedure • Wash cells or tissue twice in PBS, pellet and resuspend in 150 microliters of 0.25 M Tris HCl pH 8.0. • Freeze thaw the cells 3 times by alternating between dry ice and 37°. • Spin for 15 minutes at 4° and transfer the supernatant to a new tube and hold on ice. • Mix the following reaction solution per sample: • Incubate at 37° until a deep orange color appears. • Add 500 microliters of 1 M Na2CO3 to terminate the reaction. • Immediately read the OD574 and keep in mind that the linear range is between A574 of 0.4 and 2.7. • To compare between samples, normalize the time, volume of lysate and protein concentration. Reference: Simon and Lis (1987) NAR 15: 2971-2988. |
→如果您认为本词条还有待完善,请 编辑词条
上一篇Ultimate yeast ChIP assay 下一篇ChIP-CpG Island Microarray Binding Analysis
词条内容仅供参考,如果您需要解决具体问题
(尤其在法律、医学等领域),建议您咨询相关领域专业人士。
0