作者iamrim (笨小孩~)
看板Biotech
标题[求救] GST pull down assay 问题...
时间Thu Jul 23 00:16:18 2009
我要做protein protein direct interaction
纯化两个protein : A protein 是 small G protein(22KD)带 His tag
B protein 是 带 GST tag
A protein 纯化过程:
菌suspension在binding buffer(imidazole.tris-HCl.NaCl)+protease inhibitor
冰上sonication
Ni-NTA纯化
elution(160 mM imidazole-印象中.tris-HCl.NaCl)
a)elution的protein加入离心管(Amicon)去换成PBS,顺便想要寄降低imidazole浓度
b)没有换buffer
B protein纯化过程:
菌suspension在PBS+PI(protease inhibitor)
冰上sonication
离心,上清液和beads在4度binding 2 hrs
PBS wash 3 次,beads suspend在PBS
问题:
1.A protein与GST only beads 4度结合2 hrs,PBS+1mM DTT+1%tritonX100 wash3次
2Xsample bf去煮beads.跑胶还有A protein signal.就是negtive control做不好.
会不会是A protein 太小卡在bead,怎麽洗都洗不掉......>O<
2.有换buffer的必要吗??看别人家好像都有换.imidazole多会影响binding?
3.reduced Glutathion去elute跟直接煮bead有差吗?
感谢看完....我已经在GST pull down徘徊已久.....
希望能早日脱困~谢谢
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