s*********f
发帖数: 155 | 1 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 | 2 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 | 3 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. |
|
n***w
发帖数: 2405 | 4 Thanks. I did WB as well. Supernatants did show the specific band.
I will try freeze and thaw method rather than sonication tomorrow.
of
activity? |
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i***0
发帖数: 160 | 5 For inclusion bodies, you have to reduce temperature and IPTG amount. One of
my proteins has inclusion bodies and very small amount of soluble protein.
But when I tried with 10 uM IPTG induction, the soluble part has about equal
amount of my protein compared to inclusion bodies. But when you have
efficient cell breaking method you will release almost all of the soluble
protein from the cells. So try with more expression conditions before you
enlarge culture size. Personally, I like French Press,... 阅读全帖 |
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n***w
发帖数: 2405 | 6 Thank you guys.
I use PGEX2T vector so GST is on the N-terminus.
I normally do 100ml culture size. Here is how I did this:
1. small culture of the bacteria from glycerol stock O/N, about 6-8ml.
2. transfer the small culture to 100ml 2xYTA medium in the next day morning
, shaking at 300rpm, 37degrees.
3. it ususally takes about 1hr 40min - 2 hrs to have an OD600 around 0.75.
4. add IPTG (stock 100mM) 100ul to get a final concentration of 0.1mM.
5. Lower the temp to 22degrees and shake at 300 rmp... 阅读全帖 |
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H*g
发帖数: 2333 | 7 Probably I would do freeze-thaw for several rounds, add loading buffer, boil
for 5min, spin, and just load super to SDS-PAGE. |
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n***w
发帖数: 2405 | 8 嗯，有可能饱和了。
我昨天把1st binding后的supernatant又加beads了，看好没好些今天。
我觉得N-lau还是挺好使的。能solubilize大部分pellet。
我500ml的culture提了差不多160ug的蛋白。。。
我下次买点你说的B-Per试试好了～ （不过看成分也差不多）
我现在的protocol基本上是：(For 500ml culture)
1. thaw pellet on ice
2. add 18ml GST buffer (配方见上)，add 1% PI, add 2ml lysozyme (10mg/ml)，
mix on ice for 30 seconds
3. pass thru 20G syringe to reduce viscosity, also add DNAse 40ul (1mg/ml).
4. sonication (it will become clear)
5. 10000rpm, 40min，4deg
6. collect supernatant for GST beads binding
不过我这次还是将G... 阅读全帖 |
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p******y
发帖数: 47 | 9 谢谢楼上的。我最后都加到300UG的蛋白了，还是啥都么有~
我试试SHORTEN BOIL TIME 这次~
这个蛋白应该是能测到的，原来的AB就可以，但是原来那个FROZEN AND THAW了很多次
以后就彻底罢工了~哎 |
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r********g
发帖数: 109 | 10 FROZEN AND THAW killed the antibody. |
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h*******o
发帖数: 4884 | 11 If you are gonna use the antibody frequently in a few weeks, use Superblock
T20 or P20 from pierce to dilute the antibody and put in 4oc, it works
perfectly for a couple of weeks.
For long term use with low frequency, let's say once a month for about half
year, -20oC is OK. But freeze-thaw cycle does kill antibody significantly. |
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h*******o
发帖数: 4884 | 12 sodium Azide at low concentration mainly interferes with the peroxide
reaction (HRP conjugated 2nd antibody). It should be fine to use at a very
low concentration.
After all, it is all up to the antibody. For b-actin, sure freeze and thaw
is fine.
For some delicate antibodies, it will be a big problem.
So for me, unless it is extremely hard to get the antibody, I woudn't reuse
for more than 5 times, usually 3 times. |
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S****r
发帖数: 982 | 13 Thanks! We tried in two other cell lines, both with similar transfection
efficiency, but still can see Renilla value from 200 to 1000, while in RAW
cells, it's only around 10.
I'm also not sure if RAW is tougher to be lysed in passive lysis buffer.
Should I try freezing-thawing cycle to disrupt them?
transfection |
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s**u
发帖数: 9035 | 14 All reagents, buffer need thaw and centrifuge throughly. |
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m**a
发帖数: 1228 | 15 先试试这个
我们以前一个类似的蛋白就是用pET22b在
periplasmic表达的
纯化的时候freeze/thaw，不用破菌
bonds |
|
o*****r
发帖数: 156 | 16 EDC and NHS are aliquoted and stored at -20, every time when you need to
activate the CM chip, thaw both of them, mix and inject over the surface (I
usually use 5 ul/min for 7 min).
http://cspr.uthscsa.edu/activation.php |
|
t****g
发帖数: 120 | 17 在做inducible cell lines,要用到doxycycline，从我读到的一个protocol看
doxycycline好像很不稳定，freeze-thaw cycle不能超过７次，在medium里的half
life只有24小时； 那岂不是天天都要加新的？ 请问你们是怎么用的？--- final
concentration? frequencing of adding to medium? storage condition？
谢谢！ |
|
V***b
发帖数: 3419 | 18 20 nM is more than enough to inhibit TORC1. Stock solution 20 uM in DMSO;
make small aliquote and keep in -80; freeze-thaw cycle does not severely
impair its function, but not recommended. |
|
l**********1
发帖数: 5204 | 19 RE: 德艺双馨的楼主
please go to
//www.creativebiomart.net/description_10243_28.htm
You Position: Home >> Antibody >> Polyclonal Antibody >> Rabbit PAb >> Anti-Cdh5 PAb
Anti-Cdh5 PAb
Cat. No.
CPB-133RM
>
Immunogen
Recombinant mouse CDH5 protein.
Antibody Type
Rabbit Polyclonal Antibody.
Ig Type
Rabbit IgG.
Formulation
0.2 μm filtered solution in PBS with 5% trehalose
Preparation
Produced in rabbits immunized with purified, human cell-derived, recombinant mouse CDH5 extracellula... 阅读全帖 |
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d******1
发帖数: 709 | 20 Freeze–Thaw Cycle is a way to make protein amyloid. |
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b****s
发帖数: 148 | 21 once proteins were brewed in SDS-PAGE buffer with DTT or bME, they can be
stored at -20C and no need to be boiled again.
personally i prefer boiling samples freshly prior to gel loading.. my
samples can be stable for 1-2 months with several freeze-thaw cycles.
. |
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l**********1
发帖数: 5204 | 22 Here you go.
Casals G. et al. (2009)
Hypoxia induces B-type natriuretic peptide release in cell lines derived
from human cardiomyocytes.
Am J Physiol Heart Circ Physiol. 297:H550-5.
link:
//www.ncbi.nlm.nih.gov/pubmed/19542490
citation:
Cell culture.
AC16 cells were generated from primary human
ventricular cardiomyocytes, as previously described (7). This immortalized,
stable cell line can be repeatedly frozen, thawed, and propagated.
Cells were seeded (1 106 cells/well) in gelatin-coated six-... 阅读全帖 |
|
b******y
发帖数: 627 | 23 Upon purification, freeze in glycerol using liquid N2, store at -80, thaw it
when in use. |
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a******8
发帖数: 56 | 24 The amount of DNA is ok.
You are right that the probe is too long. Try using short and specific
probes.
Wash your membrane with more stringent buffer as there are dots on your
membrane.
Finally, genomic DNA quality can be a concern. Avoid votex during extraction
and frequent freeze/thaw cycles.
Good luck. |
|
g*********3
发帖数: 177 | 25 大家好。
最近一段时间在挣扎gateway LR reaction:
用的是pLXSH gateway 作为destination ector
用的另一个entry ector含有attL1 ATTL2
所用的质粒测序无误含有recombination site。
用的是invitrogen的LR cronase enzyme mix II.
pLXSH is resistant to Amp and After LR reaction, I do transformation. I didn
't get any colony. [Zero.]
Entry vector is amp resistant too. So I cut the amp out and linearize this
vector.
I also do LR reaction with plxsh + Gus. Gus is positive control entry vector
provided by the kit.
There is no colony.
I have done positive p... 阅读全帖 |
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c*******7
发帖数: 314 | 26 我试过直接冻－80，不过freeze and thaw至少会浪费60％，后来直接做fresh的 |
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D**R
发帖数: 371 | 27 Need to consider followings: total cell /live cell before Freeze,起始细胞不
能太少1e6-10e6 /vial，freeze protocol，直接放冰箱还是用machine protocol几分
钟降几度到-70c？ thawing protocol. |
|
a**********2
发帖数: 28 | 28 谢谢回复！
1.我有很多stable transfected的细胞，试着thaw了几瓶，大部分都活不下来，小部分
可以；实验室里没有其它人用HEK293，其它cell line例如CHO, NMuMG都很正常；
2.试过换新的FBS，也试过旧的，没什么区别；
3.应该是死了，这批是adhesion的，悬着好像不能长；
4.正在查。1月里forzen储存的细胞应该是没有。今天刚把一批悬浮死掉的细胞送出去
做mycoplasma检测。 |
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a**********2
发帖数: 28 | 29 谢谢回复！
1.我有很多stable transfected的细胞，试着thaw了几瓶，大部分都活不下来，小部分
可以；实验室里没有其它人用HEK293，其它cell line例如CHO, NMuMG都很正常；
2.试过换新的FBS，也试过旧的，没什么区别；
3.应该是死了，这批是adhesion的，悬着好像不能长；
4.正在查。1月里forzen储存的细胞应该是没有。今天刚把一批悬浮死掉的细胞送出去
做mycoplasma检测。 |
|
g*********5
发帖数: 2533 | 30 Isolation of nuclei for the co-IP and whole ChIP protocols is based on the
methods of Gendrel et al. (2002, 2005), Johnson et al. (2002), and Nelson et
al. (2006).
METHOD
For extraction and co-IP of nuclear proteins, see Steps 1-39 (Fig. 1). For
the ChIP procedure, see Steps 40-69 (Fig. 2).
Figure 1.
View larger version:
In this page
In a new window
Figure 1.
Flowchart for the timeline and organization for co-IP of nuclear proteins
from Arabidopsis seedlings.
Figure 2.
View larger versio... 阅读全帖 |
|
h***a
发帖数: 145 | 31 http://www.sciencedirect.com/science/article/pii/S0011224003000
Caspase-mediated apoptosis and cell death of rhesus macaque CD4+ T-cells due
to cryopreservation of peripheral blood mononuclear cells can be rescued by
cytokine treatment after thawing
Surojit Sarkara, Vandana Kaliab, Ronald C Montelaroa,
Thank you very much!! |
|
发帖数: 1 | 32 最近做一个DNA damage的实验，就是用H2O2来诱导Hek细胞基因组oxidative damage；
然后用FPG这种能识别8-oxoG的酶来切，被切的基因组位点会产生nick，阻碍PCR
polymerase，基于这样的原理做qPCR定量来研究某段序列对于oxidative damage的反
应。
好像这种做法比较少见，大部分DNA damage的研究都是global的。但也有很好的先例比
如：
http://www.nature.com/nature/journal/v429/n6994/abs/nature02661.html
这篇经典的Nature 文章
我按照这篇文章的做法，把genomic DNA进行FPG overnight digestion （DNA+FPG+Neb
buffer+BSA），同时也做了mock control（DNA+水+Neb buffer+BSA），然后qPCR发现
Ct如下：
DNA：25
DNA+FPG+buffer：28
DNA+水+buffer： 28
让我惊讶的是，相比于纯DNA，mock control的DNA+水+buffe... 阅读全帖 |
|
t**w
发帖数: 82 | 33 【 以下文字转载自 Postdoc 讨论区 】
发信人: thaw (Postdoc?), 信区: Postdoc
标 题: 请问大家面试的费用怎么算的？
发信站: BBS 未名空间站 (Wed Sep 16 21:53:24 2009, 美东)
老板要面，说可以负担我的费用。
大家是自己订票和旅馆还是对方给办的？ |
|
D******e
发帖数: 1085 | 34 What a douche bag if it is true. -80 will not thaw o/n. Let me test it tonite and get back to you tomorrow. Somehow, i think faint2010 is just messing with us.
of |
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a****n
发帖数: 53 | 35 我过去都是在要做反应的瓶子了，液氮freeze thaw几次，就假定干燥了吧，然后用，
做过很需要干燥的反应，还好，不知道行不行。 |
|
F**********0
发帖数: 503 | 36 I don't think it is due to liquefaction, either. Frost and thaw might be a
reason, besides that I don't think this type of soil would lost compaction
over time. |
|
S****X
发帖数: 20 | 37 By June 30, 2014, applications are invited for at least two Graduate
Research Assistant positions at the Department of Civil and Environmental
Engineering, Washington State University, Pullman (www.ce.wsu.edu).
Successful candidates will be exposed to the fields of innovative concrete
materials and cementitious composite materials. Research projects are funded
by the USDOT and State Departments of Transportation (DOTs). Likely
projects will be conducted in the Laboratory for Advanced and Sustai... 阅读全帖 |
|
t***u
发帖数: 20182 | 38 来自主题: Macromolecules版 - ATRP求助 ATRP有时候是很诡异，楼上说的对，先看看GPC和核磁吧。另外，去氧气的时候只是
freeze-purge,如果thaw一下会不会好一些。 |
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t***u
发帖数: 20182 | 39 一般freeze pump thaw 3 个cycles |
|
M*****9
发帖数: 320 | 40 N2 bubble, or freeze-pump-thaw
Either way works for me. |
|
d**a
发帖数: 3715 | 41 I recently synthesized a monomer with a styrene as one functional end group
(confirmed by NMR) and try to co-polymerize this monomer with commercially
available styrene
I ran the reaction in this way:
mix my monomer A, styrene, and AIBN in tolunene
A: 0.25 g, 0.48 mmol
styrene: 0.05 g, 0.48 mmol
AIBN 0.0055 g, 0.034 mmol, 3.5 mol% of monomers.
Freeze-thaw degas twice,nitrogen purge, in 50 mL of toluene at 80 degree C,
stirring for three days.
But it came out nothing has happened. I just checked |
|
c***r
发帖数: 4631 | 42 高手，我对RAFT有一点不懂的就是我总觉得会有一些合成出来高分子仅仅受到引发剂的
作用，没受到CTA的影响，这些高分子的pdi完全不受限制啊！怎么能减少这些高分子的
量呢？
另外合成过程对氧要求有多高呢？我看到有的就是用氮气吹20分钟，一般要freeze-
vacuum-thaw三个回合。
对水的要求呢？ |
|
b*****y
发帖数: 40 | 43 First very sorry about what happened. Hope nothing happened with the safety
of yourself.
The b.p. of EO is only ~ 10 deg C, it is extremely flammable, and it is an
carcenogen, so give it with your highest care possible. That means, no
nights, no weekends, always have labmates nearby, among many other things.
Out of the three reasons, #1 is highly possible. Definitely dilute with much
much more THF. I typically have 40 grams of EO with ~ 800 mL of THF, but at
40 deg C. If you have done a good job... 阅读全帖 |
|
b*****y
发帖数: 40 | 44 First very sorry about what happened. Hope nothing happened with the safety
of yourself.
The b.p. of EO is only ~ 10 deg C, it is extremely flammable, and it is an
carcenogen, so give it with your highest care possible. That means, no
nights, no weekends, always have labmates nearby, among many other things.
Out of the three reasons, #1 is highly possible. Definitely dilute with much
much more THF. I typically have 40 grams of EO with ~ 800 mL of THF, but at
40 deg C. If you have done a good job... 阅读全帖 |
|
i****g
发帖数: 3896 | 45 http://blog.sina.com.cn/s/blog_c24597bf0101b871.html
致谢：I would like to thank Prof. Shing-Tung Yau for suggesting the title of
this article, Prof. William Dunham for information on the history of the
Twin Prime Conjecture, Prof. Liming Ge for biographic information about
Yitang Zhang, Prof. Shiu-Yuen Cheng for pointing out the paper of
Soundararajan cited in this article, Prof. Lo Yang for information about
Chengbiao Pan quoted below, and Prof. Yuan Wang for detailed information on
result... 阅读全帖 |
|
p********y
发帖数: 5141 | 46 An interesting read on what to do when you encounter a dog while cycling.
------------------------------------------------------------------------
A few tips for dealing with pushy pooches when you’re out riding.
By Neil Bezdek
New York City has its drivers. Colorado suffers from an oxygen shortage.
Portland is a monsoon. California can’t afford to fix its potholes. New
England won’t thaw till May. The Midwest is a wind tunnel. And the
Southeast, my current training ground, has dogs. Every worko... 阅读全帖 |
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k**a
发帖数: 1124 | 47 我冻了7个，但是第三天冻的，所以都没谱儿。THAW完以后能不能活都还不知道啊。
我打算transfer 3个。
我做的是自然的，自测LH surge。总共就需要做两次u/s, 一次LH surge前，一次后。
后面那次主要是查lining等等。然后会做个blood test 查progesterone等等to
confirm ovulation did occur. 如果一切正常在LH surge后第五天transfer.
我打算用assisted hatching. |
|
k**a
发帖数: 1124 | 48 我本来是想移植三个。但礼拜六那天见到那个名大夫，他一再强调如果两个胚胎质量都
好的话，只移植两个。他还说最新guidelines的上个星期刚刚出来云云，我这个年龄的
不要超过两个。反正最终还是要看transfer那天thaw的结果啦。 |
|
k**a
发帖数: 1124 | 49 THANKS! yes, it went well. easy and smooth, just like last time. Two were
thawed and both survived. The embroyologist said they both look good. no
cells were lost. So i just transferred two with assisted hatching. |
|
m***6
发帖数: 71 | 50 刚做了thaw cycle transfer. 医生没有提要用crinone 8% 因为是周末, 联系不到医生
, 不知有没有姐妹们知道的. 可帮我解答一下. |
|
