s****9
发帖数: 932 | 1 I understand it can be the problem of the antibody and will never be solved,
but want to try as hard as possible.
Could you suggest a few antigen retrieval method? Do you think 5min acetone
fixation is enough to expose the antigen epitope?
Very few background on the staining. Do you think it is worthy to try
different blocking reagents?
Thanks so much. Really appreciate your suggestions.
or |
|
v***a
发帖数: 1242 | 2 用的reagent是可以enhance signal的那种 |
|
p*******r
发帖数: 59 | 3 Career Opportunities at JCVI Infectious Disease (8 Positions)
The PIs did not ask me to post here, I did this just in case someone is
looking for similar positions but did not see these, and I would be happy to
see some chinese fellows to come. All the PIs are very nice.
All listed at
https://careers.jcvi.org/careers/Careers.aspx
http://www.jcvi.org/
No. 1 & No. 2 Post-Doctoral Fellows
Job Responsibilities
JCVI is looking for two Post-Doctoral Fellows to join the Infectious Disease
Group in our ... 阅读全帖 |
|
v***a
发帖数: 1242 | 4 实验室只有Y-PER protein extraction reagent了，我想用它提取BL21 (DE3)产生的蛋
白，不知道可不可以啊？
谢谢！ |
|
T*****e
发帖数: 315 | 5 做了一个萤光蛋白和 tubulin 的 fusion gene，测序什么的都是对的，但是转染了之
后没有萤光信号，已经确认了
transfection reagent 什么的没有问题， linker是7个aa, 这个应该也够了，请问有
什么可能的原因造成没有信号呢？
thanks |
|
i********g
发帖数: 77 | 6 Job Description
This position is in a R&D group that manufactures reagents and instruments
for in-vitro medical diagnostics. Day to day activities will include data
analysis and process development studies to support R&D feasibility and
verification/validation project phases.
The applicant must have minimally a BS or MS in Chemistry, Biochemistry or
related discipline with experience or an advanced degree in a similar field.
Applicants with industrial background and knowledge of GMP/ISO will be
... 阅读全帖 |
|
i********g
发帖数: 77 | 7 Another one at same location:
Job Description
This position is in a group that manufactures intermediates to support
reagent manufacturing in in-vitro diagnostics. The applicant should have a
BS in Chemistry, Biochemistry or related discipline with some experience or
an advanced degree in a similar field. Applicants with some industrial
background and knowledge of GMP/ISO will be preferred.
The successful candidate will have some experience in mammalian cell culture
and hybridoma work.
Knowledge... 阅读全帖 |
|
d***s
发帖数: 1062 | 8 Sorry cannot type in Chinese.
Thank you for all your adivces. Just finished one exam. I have already
chosen another PhD PI.I like both of their researchs, so the decision is not
very tough. I just more like buying reagents freely in the MD's lab and
like his personality.Actually I think he is good mentor. Although he is very
busy with clinic jobs, he will come to the lab at least once a day,
normally twice talking about the experiment and discussing results. |
|
y*****h
发帖数: 166 | 9 Position level – Research Associate III or Senior Research Associate –
will be determined based on candidate’s background and experience.
Responsibilities and Duties
Responsible for the set up and performance of both in vitro and in vivo
experiments. The candidate will set up and perform in vitro experiments
using purified primary cells from human blood or mouse tissue. The candidate
will be involved in complex in vitro experiments requiring high technical
skills and precise planning. Addition... 阅读全帖 |
|
T**********t
发帖数: 1604 | 10 UV测蛋白浓度比BCA并不会特别不准，本质上都是吸收光谱嘛。
相反，因为UV测浓度不需要pipetting，不需要加特殊reagent，比BCA assay什么更便
捷，而且珍贵样品还可以回收。一般如果蛋白有紫外吸收，都可以用UV测浓度。
但是实际操作上，确实会有用计算出来的extinction coefficient算出来的浓度不准的
情况，这种情况大部分是因为蛋白的folding不同，有的蛋白里Trp等吸收基团都是暴露
在外面，有的蛋白中这些基团却是被包裹在蛋白内部的，所以含有同样成分的蛋白质，
有可能对紫外的吸收情况不一样。在拿到一个新蛋白时，为了确定它的extinction
coefficient，可以把这个蛋白用Guanidine HCl彻底变性，比较它变性前后的UV
absorption有没有变化，就知道计算出来的extinction coefficient是不是准确，如果
不准确的话，也可以通过这个方法算出表观的extinction coefficient应该是多少。 |
|
q*****n
发帖数: 331 | 11 For mouse experiment, you are going to need large quantity of antibodies or
small kinase inhibitors. It is unrealistic to buy them from bio-reagent
company.
what your boss can do is to set up collaborations with big people working in
the IGF field who has the antibody or inhibitor, or collaborate with
pharmaceutical company. For example:
OSI: OSI-906
BMS: BMS-754807
You can search pubmed to find references for these two drugs and author
information.
Good luck! |
|
G*****h
发帖数: 320 | 12 2-kb with 44% GC should not be too difficult. Below are what I can think of
at the moment:
1) There may be a very difficult secondary structure in the sequence (such
as an inverted repeat). If there is
indeed an inverted repeat, you have to run two separate PCR and then ligate
your two product together.
2) Some other secondary structure problems can be resolved by designing
longer primers, so you can have a
higher annealing temperature to overcome the problem.
3) Your template DNA is degraded, s... 阅读全帖 |
|
m***T
发帖数: 11058 | 13 楼上shakuras的几个贴子已经解释得挺清楚了。每个平台都有它的优缺点。Ion的优势是
simplicity, speed and scalability。说得通俗些，它的机器本身就是一台敏感度很
高的pH meter，核心技术其实都在chip里，所以它的运营成本会比较低。这些优点在
IVD方向都是比较重要的几个因素。
但它目前也有自己的缺点，比如throughput相对低，再有就是还需要emulsion PCR，都
是不利因素。好在目前整个公司对Ion项目的支持力度很大，有了invitrogen和ambion
的reagents，再加上AB这么多年在仪器方面的经验，所以Ion的前景应该还是不错的。
比如，Ion能在这么短的时间内发布OneTouch（自动化地emPCR），和整个公司对它的大
力支持是分不开的。 |
|
k****f
发帖数: 29 | 14 在测luc时，promega的protocal上说要对于96孔板来说，加75ul luciferase reagent.
但老板说太多，这个试剂贵。她说家少点，那我加20ul在75ul cell lysis 中可以吗？
试过预实验，貌似还行，但有点不确定，所以问一下有经验的大侠撒
谢谢！ |
|
c****b
发帖数: 1230 | 15 最好能用于amaxa的，他家的东西实在太tmd的贵了 |
|
n**8
发帖数: 221 | 16 请教有转染过 C2C12 和 10T 1/2 的大侠，一般分别什么转染试剂适用于这两种细胞？
不甚感激！ |
|
L**********O
发帖数: 1761 | 17 I haven't used these two cell lines, but I feel lipofectamine may work |
|
o*****r
发帖数: 156 | 18 Mirus LT-1 works really well, high efficiency (similar to lipofectamine/
lipofectamine 2000), less toxic.
I don't like the PolyFect from Qiagen. |
|
n**8
发帖数: 221 | 19 谢谢回复！
oranger大侠用Mirus LT-1转过C2C12？效果如何？ |
|
o*****r
发帖数: 156 | 20 I didn't work on C2C12 before, so I don't know about this particular cell
line.
My suggestion is to try both LT-1 and lipofectamine. |
|
n******u
发帖数: 418 | 21 只用过10T1/2
看你transfection后做什么了，luc assay的话，用ca2+-mediated transfection都可以
一般的话，用fugene6好了 |
|
v*********d
发帖数: 382 | 22 invitrogen Neon Transfection Syste
C2C12 90%+ efficiency. Should also work for 10T1/2
Best part, you can get a 2week free demo |
|
w******e
发帖数: 1187 | 23 请教：用cell-free expression synthesize peptide/protein, 要求newly synthe-
sized peptide/protein自动被immobilize到beads上，并达到尽量高的copy number。
最直接的方法大概是在gene里加个FLAG/HA的sequence，beads上coat FLAG/HA
antibody。但Ab个头太大担心copy number上不去。
请教有什么alternative method？比如his6-peptide的coverage会不会更高？
比如有没有site-specific biotinylation的reagent可以跟cell-free expression
合用？
非常感谢！ |
|
w******e
发帖数: 1187 | 24 peptide的affinity方面有新突破吗？还是大多都在uM量级？能不能推荐两篇
经典review？多谢！
另外最近多看了点东西，ms circularization对affinity的改善也有限？
看到两篇mRNA display搞出low nM peptide让我大跌眼镜，可是不知道是不是
昙花一现。有用yeast/bacteria display做peptide maturation的牛组吗？
往大点说，thermostable affinity reagent是holy grail级难题，要是peptide
不争气，岂不是始终只有aptamer一个参赛选手呵呵。指望靠avidity取胜的话，
用在in vivo/cell上还make sense，target是monovalent的话不能提高apparent
affinity吧？ |
|
w******e
发帖数: 1187 | 25 是有很多工作可以做，但如何入手就是另一回事了呵呵。
比如说，我觉得ultimate goal for affinity reagent development是thermostable,
self-integrated with detection & amplification mechanism, adaptable into
multi-target platform, tunable affinity under enviromental cue，tunable
density to achieve multivalent interaction.
那么怎么去实现这个目标？我脚的on paper aptamer其实是最好的candidate，但
过去二十年都被技术瓶颈限制着呐。最近的breakthrough能把boundary push到
多远？我没本事判断。类似的，protein engineering在thermostability上，
在integrating detection module/scaffolding module上，在molecular
switchin... 阅读全帖 |
|
w******e
发帖数: 1187 | 26 我先说个跟affinity reagent相关的：研究protein interaction和function
之间关系，最直接的技术，应该是在cell space上，限时、可逆、可控的调节
protein interaction的形成，同时观察产生的效果。现在有方法做到吗？
没有的话，what's the alternative？ |
|
L****S
发帖数: 89 | 27 Great question! I think it may be all about location. And transfection
reagents alter the distribution of ligands to different subcellular
locations.
For TLR4, it's surface vs. endosome.
For dsRNA, it's endosome vs. cytosolic.
Maybe a cell biologist can tell us how liposome tranfection works? |
|
c**g
发帖数: 14 | 28 I think your explanation of location is logical, however I have read a paper
which said that the DOTAP transfection reagents (liposome based) can
directly deliver DNA/RNA into the endosome, and the TLR3 located mainly in
the endosome,so why is RIG-1/MDA5 activated when dsRNA is transfected
through liposome? |
|
o****e
发帖数: 37 | 29 try Bio-rad's reagent. It will work much better. |
|
s**u
发帖数: 9035 | 30 All reagents, buffer need thaw and centrifuge throughly. |
|
y**u
发帖数: 7459 | 31 enzymatic assay 的 color reagent, 说 ‘measure at ex530/em580'.
microplate reader 的 wavelength Lm 该用530还是580？。。。反正我两个都测了一
遍。
谢谢！ |
|
s******n
发帖数: 47 | 32 感谢回复！
请问是用在什么细胞系
是不是用santa cruz的那个附带transfection reagent/medium转的呢？
不甚感激！ |
|
A****A
发帖数: 16 | 33 具我所知，几个fish labs正在联合用ZFN搞大规模的knock out resources。ZFN以及相
关技术最近会有比较大的进展。大家如果感兴趣，可以关注一下最近的TALEN技术。在
最近1-2年内，估计任何试验室都可以自己合成reagent去target任何基因。cost不会比
买限制性内切酶高太多。
model
transpare
rep
to
很多
serious的
大好 |
|
A****A
发帖数: 16 | 34 利用ZFN等reagents做Knock in应该是一些lab目前的奋斗方向。ZFN可以产生sequence
specific DNA double strand break. DNA双链断裂可以有效的提高homologous
recombination的频率，从而达到Knock in的目的。目前，在fish里面实现高效的HR还
很困难，即使在有ZFN的帮助下。我不是做fish的，主要做DNA repair pathway. 我觉
得可以通过一些genetic或chemical的手段抑制fish的NHEJ pathway，来增加HR的频率。
model
transpare
rep
to
很多
serious的
大好 |
|
A****A
发帖数: 16 | 35 实际上，目前大部分证据表明TALEN的off target效应要小于ZFN. TALEN现在的主要缺
点在于size太大，一个TALEN coding sequecne 至少要4kb, 而ZFN则只要1kb左右。而
且从目前以发表的和我们自己的数据来看，TALEN的剪切活性可能要低于ZFN。现在一些
实验室都在进一步优化TALEN的size以及活性，希望以后能有大幅度的提高。TALEN相对
于ZFN目前最大的优势是位点的选择范围更大。基本上每5-10bp就可以找到一个好的
TALEN位点。ZFN目前是200bp-500bp. 另外TALEN的出现打破了ZFN在genome
engineering领域的垄断地位。将大大降低reagent的价格。现在法国的Cellectis就提
供$5000TALEN的订制服务。Sigma的ZFN价格也会跟着降下来。 |
|
s******y
发帖数: 28562 | 36 transfection reagent 啊？无所谓的，各个公司的都差不多 |
|
s*******f
发帖数: 148 | 37 A recruiter sent me this email today. Not sure whether this is the right
place to post, but just would like to share it as it may be useful to some
people.
Please don't contact me. If you have questions, emailed the recruiter
directly.
=====================================================
07/18/11
Scientist (Biology)
Location - Pearl River, NY
Duration - 3 year contract
Description:
Candidates must be available for an in-person meeting with hiring manager
and team.
This is an entry-level positio... 阅读全帖 |
|
b**********8
发帖数: 349 | 38 如题，Qrt-PCR 显示细胞经某种处理后βIG-H3 mRNA有30多倍的增加，于是买回来这个
公司的抗体，按照说明书用5%BSA 按照1:1000稀释，过夜孵育后检测一点信号都没有，
即使用了很强的detecting reagents都没有。有没有正好用这个抗体的筒子，介绍点经
验吧？ |
|
e**r
发帖数: 1144 | 39 文献上看到的
测的是蛋白的荧光光谱
Emission and excitation spectra of reduced and oxidized cpYFP in the
presence
of designated reagents were obtained with a spectrofluorimeter (Model:
CM1T10I,
HORIBA Jobin Yvon, Inc.) continuously purged with nitrogen gas
他这个光谱测得到底是气相还是液相的? |
|
y*****3
发帖数: 961 | 40 Sino-American Biotech: Multiple Managerial Bioprocess Position Openings in
China for a US-China joint venture company.
The client company is an existing company well established and joint-venture
with a US Biotech company about over 10 years ago. They were very
successful in supplying Chinese market for diagnostic and reagents in the
past years. Recently they decide to expend the business into biological
product R&D and commercial manufacturing in China for both Chinese market
and global market ... 阅读全帖 |
|
o*****r
发帖数: 156 | 41 1. try to mix two plasmid at the very beginning, before lipofectamine;
2. try a different transfection reagent. |
|
F*******2
发帖数: 103 | 42 This happens a lot, first make sure your cell isin a healthy condition then
change to another transfection reagent |
|
Z******5
发帖数: 435 | 43 NE-PER® Nuclear and Cytoplasmic Extraction Reagents
Thermo （Pierce）的，货号78833或78835 |
|
|
g*****d
发帖数: 27 | 45 所有方法都试过了，hot start, redesign primers, touchdown, low primer
concentration, change all the reagents...
昨天试了最后一种方法，design tailed primers, 再不成我就崩溃了
大家有没有好的建议？ |
|
O***n
发帖数: 13127 | 46 in our lab, we do many in vivo experiments. one postdoc needs 100K a year (half salary plus benefits, half other reagents,
animal etc) |
|
f******l
发帖数: 101 | 47 我以前自己的project也是用电转做stable cell line的。
但是现在最主要是工作以后protocol不由自己选择了，二是现在是transient，电转死
的细胞太多，而普通的transfection reagent效率又太低。 |
|
s*****o
发帖数: 26 | 48 只做过一次，好像要注意不能用PBS洗细胞。
Critical Steps
Do not wash cells with a phosphate-containing buffer such as PBS. Load all
lanes of each gel with only simultaneously prepared samples. Because
transfer of phosphorylated proteins from a gel with Mn2+-Phos-tag is
difficult, the gel should be first soaked in EDTA-containing transfer buffer
B and the original semi-dry transfer method4 using three transfer buffers
should be performed.
http://www.nature.com/protocolexchange/protocols/594#/reagents |
|
s******y
发帖数: 28562 | 49 一般方法里，我用过的是lipofectamine with Plus reagent 比较好，能达到
２０％而且细胞不容易死。
效率比较高的还有另外几个，但是细胞死得很厉害，你要是无所谓的话明天我给你
找找看是哪个厂家的。
病毒转染一般能达到７０％以上 （要注意细胞浓度，最好在５０％～７５％
confluence) |
|
C****b
发帖数: 90 | 50 We have a RNAi screener who spent around 3 months to optimize the MEF
transfection condition. Finally she got 70~80% transfection efficiency.
Summary
Reagent: DharmaFECT
Cell: 30% confluent MEF instead of 70~80% confluent
Good luck! |
|
