d****i 发帖数: 2346 | 1 lysate总共也就300-500uL,除了冻干还能怎么浓缩一下? |
R******0 发帖数: 6158 | 2 有起始体积500ul的centricon,最多可以浓缩到20ul左右,不过膜本身可能吸附很多蛋
白,样品宝贵的话还是要慎重 |
Y*******n 发帖数: 15 | |
s*****g 发帖数: 7857 | 4 在国内时,拿凉氮气慢慢吹。。。体积就小了。浓缩了。
在美国,就从新提了。。。
【在 d****i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : lysate总共也就300-500uL,除了冻干还能怎么浓缩一下?
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d********n 发帖数: 15 | 5 加入10ul 4x loading bf, 然直接打开盖子放在heating blocker 90C 煮,10min后每隔
3min看一下,直到干到40ul左右,够两次 western.
(发个贴才10个WB,太少了) |
j*****n 发帖数: 217 | 6 这样蛋白会降解的
TCA是正解
每隔
【在 d********n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 加入10ul 4x loading bf, 然直接打开盖子放在heating blocker 90C 煮,10min后每隔 : 3min看一下,直到干到40ul左右,够两次 western. : (发个贴才10个WB,太少了)
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D**A 发帖数: 311 | |
s*****i 发帖数: 315 | 8 TCA 要洗干净,不然跑出来的条带很怪
【在 j*****n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 这样蛋白会降解的 : TCA是正解 : : 每隔
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T***e 发帖数: 31 | 9 可以考虑用柱子浓缩,甩一下就可以,冻干有点麻烦,还有就是蛋白会不会受到点影响?
【在 d****i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : lysate总共也就300-500uL,除了冻干还能怎么浓缩一下?
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d****i 发帖数: 2346 | 10
你是说连续加热会降解吗?还是其他原因?
【在 j*****n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 这样蛋白会降解的 : TCA是正解 : : 每隔
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d****i 发帖数: 2346 | 11
30%指什么的浓度?
【在 D**A 的大作中提到】![](/moin_static193/solenoid/img/up.png) : TCA, 保险起见,用30%
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d****i 发帖数: 2346 | 12
你的意思是用acetone多洗几遍?
【在 s*****i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : TCA 要洗干净,不然跑出来的条带很怪
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d****i 发帖数: 2346 | 13
响?
考虑过柱子,但是据说柱子对分子量不同的蛋白有选择性。
【在 T***e 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 可以考虑用柱子浓缩,甩一下就可以,冻干有点麻烦,还有就是蛋白会不会受到点影响?
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e****s 发帖数: 1125 | 14 Methanol precipitation
90%,可以再洗一次,Air dry 30-40 mins,复溶。
有效干净,复溶简单。 |
d********n 发帖数: 15 | 15 it's not degradation (proteases work less efficiently at ~90C, but will be a
disaster at ~55C at which proteases have reduced activity but protein
substrates begin to open structure and give much higher accessibility), but
precipitation because proteins lose native structure under heating. This is
why SDS or LDS loading bf needs to be added before heating. 1g sds binds to
14g proteins, 10ul sds loading bf is supposed to be enough wrap all proteins
in
the sample as they are so low.
the only problem of heating is that the salts will also be concentrated,
which will affect protein migration in the sds-page.
tca might not work well when proteins are so low, which is a common problems
for any ogranic solution-based precipitation. Besides, it needs extra step
to adjust
ph, but it might be worth a try.
A column can also be used, e.g., YM-30. but use loading bf go through the
column first to saturate/block the filter membrane, and also don't forget
use h2o to wash it so as to get rid of extra sds that forms micelle that can
potentially block the flow.
pls let us know which method works well for you. we can also learn more by
reading your experience.Thanks!
【在 j*****n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 这样蛋白会降解的 : TCA是正解 : : 每隔
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d****i 发帖数: 2346 | 16
90%是指90%回收率吗?
这个完了以后跑电泳会不会有奇怪的条带?
【在 e****s 的大作中提到】![](/moin_static193/solenoid/img/up.png) : Methanol precipitation : 90%,可以再洗一次,Air dry 30-40 mins,复溶。 : 有效干净,复溶简单。
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e****s 发帖数: 1125 | 17 90% methanol浓度。
甲醇挥发很快,不会影响。
【在 d****i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : 90%是指90%回收率吗? : 这个完了以后跑电泳会不会有奇怪的条带?
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d****i 发帖数: 2346 | 18
a
but
is
to
proteins
我的buffer里还有2%的SDS应该问题不大。非常感谢你讲的这么详细!
【在 d********n 的大作中提到】![](/moin_static193/solenoid/img/up.png) : it's not degradation (proteases work less efficiently at ~90C, but will be a : disaster at ~55C at which proteases have reduced activity but protein : substrates begin to open structure and give much higher accessibility), but : precipitation because proteins lose native structure under heating. This is : why SDS or LDS loading bf needs to be added before heating. 1g sds binds to : 14g proteins, 10ul sds loading bf is supposed to be enough wrap all proteins : in : the sample as they are so low. : the only problem of heating is that the salts will also be concentrated, : which will affect protein migration in the sds-page.
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d****i 发帖数: 2346 | 19
http://yeaki.org/index.php?title=Methanol_Precipitation_of_Prot
这个protocol用的是100%的甲醇,我打算试一试看。谢谢!
【在 e****s 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 90% methanol浓度。 : 甲醇挥发很快,不会影响。
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e****s 发帖数: 1125 | 20 一个意思。我也是用100%,但最终甲醇浓度是90%,要有样品体积的啊。
【在 d****i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : : http://yeaki.org/index.php?title=Methanol_Precipitation_of_Prot : 这个protocol用的是100%的甲醇,我打算试一试看。谢谢!
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d****i 发帖数: 2346 | 21
嗯,明白。非常感谢!
【在 e****s 的大作中提到】![](/moin_static193/solenoid/img/up.png) : 一个意思。我也是用100%,但最终甲醇浓度是90%,要有样品体积的啊。
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s******s 发帖数: 13035 | 22 找个分子量的柱子,比如3k啥的,离心个半小时就好了。
或者dialysis换buffer以后冻干,直接冻盐浓度也大了
【在 d****i 的大作中提到】![](/moin_static193/solenoid/img/up.png) : lysate总共也就300-500uL,除了冻干还能怎么浓缩一下?
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