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biotin-pulldown

Prof. Chang:
Click reaction with biotin-azide for 30 min at 37 °C, respectively. The final concentrations of each component were: 1% SDS, 5% t–BuOH, 200 µM biotin-azide, 2 mM TCEP, 0.9 mM CuSO4 and 0.1 mM Cu(TBTA). The lysate proteins were precipitated by adding 4 volumes of EtOH pre-chilled at −20 °C (EtOH final concentration is 80 % v/v). The sample was vortexed briefly and incubated at −80 °C overnight to facilitate protein precipitation. The precipitate was collected by centrifugation at 21,000 x g for 30 min at 4 °C and washed twice with pre-chilled EtOH (80%), once with pre-chilled acetone. The pellet was air-dried, then dissolved in 20–50 µL 50 mM HEPES (pH 7.6), 4% LDS and 1 mM EDTA and dissolved by vortexing and heating at 50-60 °C until the protein pellet dissolved. LDS was diluted to a final concentration of 0.5% with 50 mM HEPES (pH 7.6) and added to 50 µL of Streptavidin sepharose beads pre-equilibrated with 50 mM HEPES (pH 7.6) and 0.5% LDS. Small potion of samples was kept as input, The rest sample was incubated with beads for 2 h at room temperature by end-over-end rotation after that supernatant was removed by centrifugation at 500 x g for 3 min. The beads were washed three times with 500 µL of 50 mM HEPES (pH 7.6) with 0.5% LDS with end-over-end rotation at room temperature for 30 min during each wash. The bound protein was eluted by boiling the beads at 98 °C for 10 min with 30 µL of 2 X Laemmeli dye containing 6% βME. The sample was subjected to SDS-PAGE and then colloidal coomassie staining.
for 1-2mg lysates

my personal version:
for 1000μg lysates

  1. 收集细胞并提取蛋白以后, BCA测定蛋白含量
  2. 配置10x click mix, 10mM CuSO4, 10mM TCEP, 1000μM TBTA, 2000μM biotin-azide
  3. 细胞蛋白提取液中加入10x click mix, incubate for 1h at 37°C
  4. 加入4倍体积-20°C预冷的乙醇, 短暂vortex以后, 放至-80°C过夜, 以沉淀蛋白
  5. 离心15800xg, 30min, 4°C
  6. -20°C预冷的EtOH/H2O (80:20) 洗两次
  7. -20°C预冷的Acetone洗一次
  8. 晾干
  9. 加入30μL bufferA(50mM HEPES, 4% LDS, 1mM EDTA), 加热到50°C并振荡直至蛋白完全溶解
  10. 用 50 mM HEPES（pH 7.6）将 样品中的LDS 稀释至 0.5% 的最终浓度(保留一部分作为input检验pulldown之前也是有蛋白的)
  11. 然后加入 40 µL 用 50 mM HEPES（pH 7.6）和 0.5% LDS 预平衡过的 Streptavidin sepharose 珠中。
    1. 商品平衡至室温
    2. 混合均匀后取所需量的加入ep管中, (用剪去尖头的枪头吸)
    3. 500g离心1分钟, 去除储存液, 不要吸到树脂
    4. 加入一倍体积的50 mM HEPES（pH 7.6）和 0.5% LDS, 混匀, 500g离心1分钟, 移去上清, 不要吸到树脂
    5. 重复上步一次
  12. 室温下颠倒混匀旋转2h
  13. 500g离心3分钟, 去上清
  14. 加入500μL 50 mM HEPES (pH 7.6) 和 0.5% LDS, 室温下颠倒混匀旋转30min, 500g离心3分钟, 去上清
  15. 重复步骤13共三次
  16. 加还原型loading buffer煮10min

30μL bufferA溶解 40μL beads 30μL 3x loading 煮两次 95°C 15min, 转移两次上清