H*g
发帖数: 2333 | 1 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! |
s*******e
发帖数: 1010 | 2 You should try at least one lower temp, since sometimes it helps expression
for toxic protein. |
T**********t
发帖数: 1604 | 3 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. |
g*********r
发帖数: 9366 | 4 yes, try low IPTG conc. even down to 0.1 mM
and try room temp inducing and growing aftrwards
(
【在 T**********t 的大作中提到】
: 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.
|
n***w
发帖数: 2405 | 5 question 1: are you sure your sequence is right? ORF is not shifted?
question 2: are your product toxic to the cells? If they are, you need to
change an expression system.
question 3: is there any codon bias? If there is codon bias the expression
may be poor.
question 4: if the answer to Q1 is yes, to Q2 is no, to Q3 is no, then how
long is your induction? I have been expressing a fusion protein with Mw
116kD (including GST), I did a 4, 6, 8 hour time course and you should
ensure your induction time is enough.
question 5: if question 1-4 are not a problem to you, as they have suggested,
try lower temperature. I do all my expression under 22 degrees (I want to
try 18 degrees but my thermoshaker sucks...)
question 6: how do you determine if there is no induced expression? simply
by taking 20ul at each time points for SDS-PAGE or you centrifuge, sonicate,
etc?
Good luck. |
I***a
发帖数: 13467 | 6 检查密码子，
有的要用codon plus的菌株表达 |
X******n
发帖数: 914 | 7 Try 200 uM of IPTG at 16oC overnight. |
s*******e
发帖数: 1010 | 8 20C or even lower will be better -- 33C may not make big difference
(
【在 T**********t 的大作中提到】
: 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.
|
H*g
发帖数: 2333 | 9 Thank you, guys!
I sequenced my expression plasmid and I am sure it is correct, no frame
shift, no mutations. I feel my protein
is not toxic to E.coli. I shaked the E.coli culture at 37C till OD600=0.6,
then add IPTG to 1mM. I also had a
second culture but not add IPTG.
I took 1.5mL every hour for 7 hours from both cultures (with and without IPTG), spin
down, sonicated, and loaded side-
by-side on SDS-PAGE, I couldn't see any band being induced.
I would try lower temperature and lower concentration of IPTG today.
Thank you guys! |
p**u
发帖数: 138 | 10 I know a friend who met similar problem (tried strain for codon bias, toxic
protein, none of them worked). He deleted about 10 amino acid at the N-
terminus of the protein, then the protein was expressed well in BL21 (DE3). |
|
|
j*****y
发帖数: 94 | 11 跟风问一下，我现在也有同样的问题。DNA sequence没问题，也是BL21(DE3)细胞， 可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（25）或者更低（17）incubate 过夜？ |
T**********t
发帖数: 1604 | 12 你有做positive control的plate么？如果pos control plate上也没有或者只有很少的
菌落，就很可能是transformation的温度或者时间出了问题。否则的话，有可能你
transform的时候用的质粒量太少，或者你的蛋白有毒性。
可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（
25）或者更低（17）incubate 过夜？
【在 j*****y 的大作中提到】
: 跟风问一下，我现在也有同样的问题。DNA sequence没问题，也是BL21(DE3)细胞， 可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（25）或者更低（17）incubate 过夜？
|
n***w
发帖数: 2405 | 13 How do you do transformation?
using ligation product or the sequenced plasmid?
if you use sequenced plasmid and the concentration is around 200 ng/ul, you
simply can do a quick transformation (1ul DNA + 50ul competent cells (or
even 20ul), 20 minute on ice, 45 seconds 42 degree heat shock, 2 minute on
ice, then plate all of them).
and always set up controls.
可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（
25）或者更低（17）incubate 过夜？
【在 j*****y 的大作中提到】
: 跟风问一下，我现在也有同样的问题。DNA sequence没问题，也是BL21(DE3)细胞， 可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（25）或者更低（17）incubate 过夜？
|
j*****y
发帖数: 94 | 14 positive control plate 上有菌落的，我transform了50ng 的DNA，温度42,45sec，我
怀疑是蛋白有毒性。如果真的是有毒性，我能不能加大DNA的量？
【在 T**********t 的大作中提到】
: 你有做positive control的plate么？如果pos control plate上也没有或者只有很少的
: 菌落，就很可能是transformation的温度或者时间出了问题。否则的话，有可能你
: transform的时候用的质粒量太少，或者你的蛋白有毒性。
:
: 可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（
: 25）或者更低（17）incubate 过夜？
|
j*****y
发帖数: 94 | 15 是sequenced plasmid，我稀释了DNA到25ng/uL，然后transform了2uL到80ul的细胞里
，30min on ice，42 degree for 45 sec，plate all，没有菌落。。。
you
【在 n***w 的大作中提到】
: How do you do transformation?
: using ligation product or the sequenced plasmid?
: if you use sequenced plasmid and the concentration is around 200 ng/ul, you
: simply can do a quick transformation (1ul DNA + 50ul competent cells (or
: even 20ul), 20 minute on ice, 45 seconds 42 degree heat shock, 2 minute on
: ice, then plate all of them).
: and always set up controls.
:
: 可是transformation以后plate上没有菌落。这种情况应该怎么处理呢，是放在室温（
: 25）或者更低（17）incubate 过夜？
|
n***w
发帖数: 2405 | 16 What's the concentration of the plasmid?
I assume 25ng/ul is quite low for miniprep plasmid. And you just juse 50ng
DNA and 80ul bacteria (that's a lot for transformation). You know, heat
shock transformation efficiency is low and by doing so you make the
efficiency much lower...
I suggest you to try high concentration, let's say, 200ng/ul, and then mix
with 40ul competent cells, 30min on ice, 42 for 45s, 2min on ice and then
plate all. Let's see if there is any colony after the second try.
However, I cannot exclude the toxicity of your gene.
【在 j*****y 的大作中提到】
: 是sequenced plasmid，我稀释了DNA到25ng/uL，然后transform了2uL到80ul的细胞里
: ，30min on ice，42 degree for 45 sec，plate all，没有菌落。。。
:
: you
|
p********o
发帖数: 29 | 17 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 |
s*******e
发帖数: 1010 | 18 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
【在 H*g 的大作中提到】
: Thank you, guys!
: I sequenced my expression plasmid and I am sure it is correct, no frame
: shift, no mutations. I feel my protein
: is not toxic to E.coli. I shaked the E.coli culture at 37C till OD600=0.6,
: then add IPTG to 1mM. I also had a
: second culture but not add IPTG.
: I took 1.5mL every hour for 7 hours from both cultures (with and without IPTG), spin
: down, sonicated, and loaded side-
: by-side on SDS-PAGE, I couldn't see any band being induced.
: I would try lower temperature and lower concentration of IPTG today.
|
e****s
发帖数: 1125 | 19 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
【在 H*g 的大作中提到】
: Thank you, guys!
: I sequenced my expression plasmid and I am sure it is correct, no frame
: shift, no mutations. I feel my protein
: is not toxic to E.coli. I shaked the E.coli culture at 37C till OD600=0.6,
: then add IPTG to 1mM. I also had a
: second culture but not add IPTG.
: I took 1.5mL every hour for 7 hours from both cultures (with and without IPTG), spin
: down, sonicated, and loaded side-
: by-side on SDS-PAGE, I couldn't see any band being induced.
: I would try lower temperature and lower concentration of IPTG today.
|
H*g
发帖数: 2333 | 20 I used only supernatant this time. I also saved precipitate and haven't
tested
yet. I only ran supernatant with SDS-PAGE since I sincerely hope I could
observe a huge band somewhere, however, I didn't.
I used Invitrogen SimpleBlue (same as CommassieBlue) and haven't tried WB.
I will run SDS-PAGE precipitate as well (such as to add 50uL loading buffer,
boil and load 20 uL onto each lane).
Thanks a lot and let's go SUNNY!
【在 e****s 的大作中提到】
: 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
|
|
|
H*g
发帖数: 2333 | 21 I do have plasmids that encode different truncated form of my protein.
I would try with those, thanks a lot!
toxic
【在 p**u 的大作中提到】
: I know a friend who met similar problem (tried strain for codon bias, toxic
: protein, none of them worked). He deleted about 10 amino acid at the N-
: terminus of the protein, then the protein was expressed well in BL21 (DE3).
|
n***w
发帖数: 2405 | 22 prepare samples from whole cell lysate, pre-sonication, post-sonication,
supernatant, pellet. |
s********n
发帖数: 2939 | 23 Re this.
补充一点就是，如果你的蛋白有活性的话，你可以试试检测一下cell lysate的活性，
有时候蛋白表达了，但是表达量很少或者大部分形成inclusion body了，在SDS-PAGE
gel上根本看不出来。 |
n***w
发帖数: 2405 | 24 我现在就怀疑我的蛋白在inclusion body里面，有什么好办法解决吗？
谢谢。
【在 s********n 的大作中提到】
: Re this.
: 补充一点就是，如果你的蛋白有活性的话，你可以试试检测一下cell lysate的活性，
: 有时候蛋白表达了，但是表达量很少或者大部分形成inclusion body了，在SDS-PAGE
: gel上根本看不出来。
|
s********n
发帖数: 2939 | 25 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.
【在 n***w 的大作中提到】
: 我现在就怀疑我的蛋白在inclusion body里面，有什么好办法解决吗？
: 谢谢。
|
H*g
发帖数: 2333 | 26 Do you mean whole lysate by just adding loading buffer (let's say 50uL to
precipitated E.coli from 1mL culture), boiling for 5min, and loading 15uL to
each lane on SDS-PAGE?
【在 n***w 的大作中提到】
: prepare samples from whole cell lysate, pre-sonication, post-sonication,
: supernatant, pellet.
|
s*********f
发帖数: 155 | 27 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. |
s*********f
发帖数: 155 | 28 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. |
s*********f
发帖数: 155 | 29 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. |
e****s
发帖数: 1125 | 30 难道现在回帖都要高喊一声Sunny?! hehe
不知道别的地方，我们实验室做Expression test的标准程序是lysate和Sup都要检测，
用WB。特别要是用E.coli来表达真核蛋白，很难在考染里看到Overexpress的条带。
建议直接做WB，其实是省时间的。
buffer,
【在 H*g 的大作中提到】
: I used only supernatant this time. I also saved precipitate and haven't
: tested
: yet. I only ran supernatant with SDS-PAGE since I sincerely hope I could
: observe a huge band somewhere, however, I didn't.
: I used Invitrogen SimpleBlue (same as CommassieBlue) and haven't tried WB.
: I will run SDS-PAGE precipitate as well (such as to add 50uL loading buffer,
: boil and load 20 uL onto each lane).
: Thanks a lot and let's go SUNNY!
|
