j*****a
发帖数: 92 | 1 阴离子交换色谱 Q Sepharose Fast Flow (GE Healthcare Life Sciences)pH
stability Working Range 2-12 .用1 M NaOH 洗,用纯水洗到pH 7, 换TrisHCl
buffer pH 5.5 平衡, elute pH 变成9.洗了一天还是pH 9. 有人遇到过吗？ |
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j*****a
发帖数: 92 | 2 阴离子交换色谱 Q Sepharose Fast Flow (GE Healthcare Life Sciences)pH
stability Working Range 2-12 .用1 M NaOH 洗,用纯水洗到pH 7, 换TrisHCl
buffer pH 5.5 平衡, elute pH 变成9.洗了一天还是pH 9. 有人遇到过吗？ |
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s********n
发帖数: 2939 | 3 You column is stable between 2-12. However, you washed it by 1 M NaOH?!
And why did you use Tris-HCl for pH 5.5? Tris-HCl also works between 7-9.
Did you measure the pH offline or online? If online, did you calibrate the
pH sensor? |
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j*****a
发帖数: 92 | 4 Good point. I need to switch buffer system. |
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N2
发帖数: 81 | 5 不要被误导, 是要用强碱强酸洗的,等会有空写给你protocol |
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r***e
发帖数: 2539 | 6 顶这个，没错。
其实都不要digitonin，直接上低渗buffer，我用0.1M TrisHCl, no salt。冰上放10分
钟。10万g离心，上清就是cytosol，pellet用含1% Triton 的buffer弄出membrane pro
tein。 |
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h*****t
发帖数: 1226 | 7 1M NaOH的pH应该是14.
还有Tris buffer在低于pH6.5的时候Buffer capacity很小了. |
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k******e
发帖数: 8870 | 8 换强一点浓度高一点的BUFFER先洗洗再换成你要的BUFFER |
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