P******n
发帖数: 20 | 1 Compare uninduced and induced cells, and you should know if the protein is
overexpressed. |
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m**z
发帖数: 787 | 2 re this. And I don't think you would really need western blot to see protein
expression if the protein is overexpressed. |
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n***w
发帖数: 2405 | 3 Thank you. I did this just on SDS-PAGE but no difference. I cannot see a
difference... =(
protein |
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n***w
发帖数: 2405 | 5 我觉得我可能找到原因了。
我觉得是载体的原因。我会换载体。 |
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x*****o
发帖数: 441 | 6 提纯细胞,IPTG induce 之后5小时生长,之后就是离心.我的问题是离心之后大家都怎
么resuspend 细胞. 我同事说用磁子,搅拌.不过她说这个她自己发明的,别人都是冷室
里面用移液管上下吸溶液resuspend 细胞.她嫌太慢,就用磁子,快.
用磁子和用移液管,哪个好呢?
谢谢! |
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h**********8
发帖数: 650 | 7 你的问题我遇到过，几乎一模一样。
用的rossetta, 换了个培养基2yt，温度降到20度， iptg 降到0.2, ok 了。 |
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F*******2
发帖数: 103 | 8 想把蛋白序列克隆到一个有his-tag，有tev site, 有t7-promoter，最好还是iptg
inducible expression的vector上，看了invitrogen有合适的TOPO-TA-vector，但是好
贵啊，大家有什么推荐不？ |
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T**********t
发帖数: 1604 | 9 Of course make glycerol stocks for those strains.
It's usually recommended to renew those stocks every 6 months. But I've used
frozen stocks of several years old and still got satisfactory expression. I
guess it may vary from case to case, though.
I would do small scale expression test each time before a large scale
expression culture if I'm worried about the quality of the frozen stock.
Just use a 5-ml culture and induce as how you would with a 1-L or larger
culture (scale down the amount of IP... 阅读全帖 |
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n***w
发帖数: 2405 | 10 Actually many protocols say high concentrations of IPTG can be used at
screening stage, up to 1mM.
used
I |
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H*g
发帖数: 2333 | 11 Sorry I couldn't type in Chinese with lab computers.
I am trying to express my protein in E.coli these days. I tried with my
construct in E.coli BL21(DE3) with 1mM
IPTG at 37C for several time points, but couldn't observe any induction.
Does this mean there is no need for
me to try any lower temperature, such as 30C, 25C,etc, since even 37C has
no induction and they all won't
have any induction as well?
Thanks! |
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T**********t
发帖数: 1604 | 12 I would try again with lower IPTG conc. (say, 0.4mM) and lower temperature (
say, 33C).
Also, if your protein is toxic to E.coli cells, it helps if you use the
strain BL21(DE3)pLysS. |
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g*********r
发帖数: 9366 | 13 yes, try low IPTG conc. even down to 0.1 mM
and try room temp inducing and growing aftrwards
( |
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X******n
发帖数: 914 | 14 Try 200 uM of IPTG at 16oC overnight. |
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p********o
发帖数: 29 | 15 先把所有的密码子换成E.coli的偏好密码子，重新合成基因片段
把IPTG从0.05mM起做浓度梯度，应该没有问题，我也有类似的经历后来就这样解决了 |
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s*******e
发帖数: 1010 | 16 Do u have a cleavable His-tag on you protein? Sometimes protein (esp
membrane protein) has a low yield which can not be found by comparing
induced and non-induced samples.
If your protein is really important for you, try add a Thrombin, TEV or SUMO
protease cutable His-tag and do a simple separation with Nikel beads. Then
check if there is difference between SDS-PAGEs with and without protease
incubation. Good luck.
IPTG), spin |
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e****s
发帖数: 1125 | 17 2个问题，
1）你检测的是Supernatant还是Total lysate?
2)你是依靠考染对比还是用的Western?
如果你上样的是total lysate，然后Western看不到明显的表达，那就不用试其他条件
了。
如果你只是SDS-PAGE后，用考染，很可能有表达，但灵敏度不够，建议WB对比。
frame
OD600=0.6,
without IPTG), spin |
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s********n
发帖数: 2939 | 18 你表达的蛋白有酶活性么，有的话测一下上清，没有的话只能做WB。如果确定上清里有
你的蛋白，而且你需要的量不大，你可以试试直接从上清纯化。如果要的量较大，可能
就需要摸条件了，比如温度，IPTG浓度，strain等，实在不行再试试refolding from
inclusion body. |
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s*********f
发帖数: 155 | 19 also make sure your IPTG is good, e.g., freshly made solution, not a
solution that has been frozen and thawed multiple times, and known to induce
protein expression for other plasmids in e.coli. |
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s*********f
发帖数: 155 | 20 also make sure your IPTG is good, e.g., freshly made solution, not a
solution that has been frozen and thawed multiple times, and known to induce
protein expression for other plasmids in e.coli. |
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s*********f
发帖数: 155 | 21 also make sure your IPTG is good, e.g., freshly made solution, not a
solution that has been frozen and thawed multiple times, and known to induce
protein expression for other plasmids in e.coli. |
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n***w
发帖数: 2405 | 22 Hi, all,
I am using Rosetta-Gami DE3 pLySs to expression my fusion protein which is
predicted as 91kDa.
I first did the expression assay and found protein expression was induced
after adding IPTG by testing the whole cell lysate. (bacteria at certain
time points, add 2X sample buffer, boil, SDS-PAGE).
Then I moved to culture more and lysed the cells using a sonicator, after
resuspending the pellet with 1x cold PBS/1% PI. After all these, I loaded
the supernatant and pellet side by side and stain... 阅读全帖 |
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g*********r
发帖数: 9366 | 23 这一步要WB和commasie同时跑
大多数inclusion body的蛋白在WB 上也会显现，这个没什么意义
要看在supernatant 中有多少
而且要充分离心，取真正的清液
办法， 降温，少加IPTG，等等
实在不行denature |
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g*********r
发帖数: 281 | 24 Maybe it is not necessary to ask, where did you put your GST? Normally N-
terminal GST will help your protein folding and solubility.
In term of inclusion body, like people mentioned, lower temperature and IPTG
concentration might help.
Or try some other Tag, like MBP. |
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n***w
发帖数: 2405 | 25 Hi, how much did you inoculate into the final culture? 0.1mM IPTG is the
final concentration or the stock concentration? Thanks!
0,
300rpm |
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w*********e
发帖数: 98 | 26 I inoculated 5ml overnight culture (37 C)into 30ml medium because I was
using 18 C to shake ( cell grows much slower than 37C) until OD is 0.8 and
then I added IPTG at final 0.1mM. The speed of shaker when I started using
18 C is 350 rpm.
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w*********e
发帖数: 98 | 27 I used 30ml because I was trying to see if my protein could be expressed.
After IPTG was added, the culture was shaked at 18 C for 20 hours in order
to get enough cells.
The big difference based on your protocol is that I started low temperature
the next morning when I did final inoculation, while you still used 37 C.
I compared the soluble and the pellet fractions at 18, 28 and 37 C,so I know
18 C is the best one. |
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d***y
发帖数: 8536 | 28 发酵过程中加那么多IPTG，这价格就不可能太便宜。 |
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x********u
发帖数: 430 | 29 The only downside of large-scale fermentation is the energy input.
For a typical 30,000L aerobic fermenter, the motor (agitator) will consume
more than a few hundred thousand Watts, not to mention the downstream
purification process.
IPTG is prohibitivly expensive to be used for large scale fermentaion, some
alternative inducers such as lactose or arabinose will be used. |
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d***y
发帖数: 8536 | 30 发酵过程中加那么多IPTG，这价格就不可能太便宜。 |
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x********u
发帖数: 430 | 31 The only downside of large-scale fermentation is the energy input.
For a typical 30,000L aerobic fermenter, the motor (agitator) will consume
more than a few hundred thousand Watts, not to mention the downstream
purification process.
IPTG is prohibitivly expensive to be used for large scale fermentaion, some
alternative inducers such as lactose or arabinose will be used. |
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s**********y
发帖数: 23 | 32 基因大小 2.7Kb, 蛋白质大小约为130KD，IPTG诱导3h，SDS-PAGE胶中大部分蛋白集中
在60KD附
近，目标蛋白似乎有，但是相对于同期的GST实在很少。
目前猜测是因为蛋白太大，大部分E.coli未转录完全。
本人大四，在做毕设。。没有什么经验，想请教下有没有其他的可能原因，还有，该怎
么改善下！ |
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s******y
发帖数: 28562 | 33 Research Products International Corp 的又便宜又好使。5克只要48美元 |
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M*****n
发帖数: 16729 | 34 多买点，好像还送BlueRay DVD player呢。 |
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n*********m
发帖数: 38 | 38 Check Goldbio. It's really cheap. they even provide 1gm free sample |
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g*********r
发帖数: 281 | 39 记得在国内买的都是一克20多人民币，也都号称是进口分装的。现在怎么都见不着这么
便宜的呢。是不是以前那些都是骗人的呀？还有agarose这种东西，在国内号称进口的也好便宜啊，这里怎么就那么死贵死贵的。看来也是骗人的。 |
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v***a
发帖数: 1242 | 41 是不是可以直接把有bacteria的LB进行离心，然后往pellet里加SDS Sample Buffer后
煮沸3分钟即可上样跑胶？
谢谢！ |
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m**z
发帖数: 787 | 42 yes. i usually boil for 10min |
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v***a
发帖数: 1242 | 44 谢谢。SDS Sample Buffer你是用4X的吗？ |
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v***a
发帖数: 1242 | 45 我还不知道这个蛋白表达量多不多，所以先试试。如果低的话，怎么做比较好？
多谢！ |
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W*******a
发帖数: 1769 | 48 western against the tag |
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v***a
发帖数: 1242 | 49 多谢提醒。开始只想到了用Coomassie gel。 |
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s********n
发帖数: 2939 | 50 我建议你还可以保留一部分样品，做supernatant和precipitation的比较，可以看出是
否是soluble expression。 |
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