s******y 发帖数: 28562 | 1 OK, it's call Millipore* Centriprep* Centrifugal Filter Units
They have 30 kD cut off but can only hold 15mL sulotion.
the
inserted |
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C*******e 发帖数: 4348 | 2 谢谢sunnyday,还特意去帮我查了产品
怎么说呢,这种centrifugal filter unit我很怀疑会很快就堵住了
尤其像lysate这种蛋白浓度很高的东西
我就是想知道有没有办法粗分一下
事实是这样的,GST-tag表达蛋白以后有一多半都是GST(25 kDa)
比较少的部分是GST-fusion protein (50 kDa)
纯化的时候GST绑在柱子上
得到的目的蛋白只有一点点(1 liter culture得到300 ul * 1 mg/ml)
这个好像是个常见问题
好像有的可以通过额外连一段linker来解决
我就是想在不重新做construct的情况下
如果有办法可以一开始粗分一下,然后再上柱子,就方便多了
所以想的是有没有什么原始的生化方法可以粗分的 |
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s******r 发帖数: 2876 | 3 能不能用硫酸铵沉淀?
重做constructs显然要容易得多。
谢谢sunnyday,还特意去帮我查了产品
怎么说呢,这种centrifugal filter unit我很怀疑会很快就堵住了
尤其像lysate这种蛋白浓度很高的东西
我就是想知道有没有办法粗分一下
事实是这样的,GST-tag表达蛋白以后有一多半都是GST(25 kDa)
比较少的部分是GST-fusion protein (50 kDa)
纯化的时候GST绑在柱子上
得到的目的蛋白只有一点点(1 liter culture得到300 ul * 1 mg/ml)
这个好像是个常见问题
好像有的可以通过额外连一段linker来解决
我就是想在不重新做construct的情况下
如果有办法可以一开始粗分一下,然后再上柱子,就方便多了
所以想的是有没有什么原始的生化方法可以粗分的 |
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m*********7 发帖数: 5207 | 4 Maybe you can filter your lysate using 0.22um filtration unit, or centrifuge
at high speed to get rid of precipitates/protein aggregates first. |
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T**********t 发帖数: 1604 | 5 50ml的lysate直接过0.22um的filter的话,绝对是会堵的。我那时是用syringe拿手压
的,连换了三个filter才算搞定,累了个半死。如果用vacuum可能会容易一点吧。
centrifuge |
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i******m 发帖数: 495 | 6 just a little bit of glass fiber can do that (along with centrifugation) |
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s******y 发帖数: 28562 | 7 不知道为什么国内没有扶持起来一个两个这个方面的大公司?按说这个技术也不是
很难的呀。比方说那些个micro-centrifuge tube, 国内那些真的就是盖子是盖
不牢的,如果盛的东西多一点,一离心居然会飞出很多溶液来!
国内某些高校办的什么所谓高科技公司那么盛行,为什么就没有人办这种公司? |
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l*******k 发帖数: 361 | 8 我以前老板的,
Everyone will die, why do you need to worry about your parents?
请假回国带父母看身体的时候
You didt make it work.
全世界没有人做出来过的实验,而且我就不知道什么实验是你可以make it work
Have you spent any time to improve your English?
English也成了不让人毕业的理由
It is your karma
在他手下干了四年,发了三篇paper却不能毕业
Why dont you do something else during centrifuge? Why do you want a PhD
degree? Why dont you get a master degree and get out here?
PhD第五年,某天周日下午做实验等离心的十分钟里面 |
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n*****s 发帖数: 8 | 9 I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
asked to do a second precipitation after the elution step in order to
further remove the salt contamination. I use -20C isoproponal centrifuge at
room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
the DNA in EB buffer.
I did not see the precipitation at the bottom of the 2ml tube after the 30
min, a senior phd student told me that sometimes even one can not see the
precipitation one will still get so... 阅读全帖 |
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m*****y 发帖数: 857 | 10 in most cases, you can see the precipitate from IPP centrifugation.
precipitate may be invisible from 70% EtOH precipitate though
at |
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s******r 发帖数: 2876 | 11 The bacteria may be over grow,
try to pick up a new colony.
I used Qiagen plasmid plus Maxiprep kit to purify my construct. And I was
asked to do a second precipitation after the elution step in order to
further remove the salt contamination. I use -20C isoproponal centrifuge at
room temp for 30 min, 15000xg, then wash with 70% ethanol, and resuspend
the DNA in EB buffer.
I did not see the precipitation at the bottom of the 2ml tube after the 30
min, a senior phd student told me that sometimes... 阅读全帖 |
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d********r 发帖数: 3279 | 12 Arsenic-associated bacteria (NASA's claims)
ResearchBlogging.org
Wolfe-Simon F, Blum JS, Kulp TR, Gordon GW, Hoeft SE, Pett-Ridge J, Stolz JF
, Webb SM, Weber PK, Davies PC, Anbar AD, & Oremland RS (2010). A Bacterium
That Can Grow by Using Arsenic Instead of Phosphorus. Science (New York, N.Y
.) PMID: 21127214
Note to new readers: I wrote this post on Saturday Dec. 4, mainly to
clarify my own thinking. I didn't expect anyone other than a few
researchers to ever read it. Since then I've made ... 阅读全帖 |
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C******8 发帖数: 602 | 14 最近一段时间长这个cell总会发现media里脏乎乎的,有一些不知道是deris还是啥,
每次多用1xpbs多洗两遍也不行。。。
倒应该真不是mycoplasma contamination,因为前几天测还是negative的。。。
和以前的subculture也没多大区别啊。。。1200rpm5分钟centrifuge的 |
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r******y 发帖数: 21907 | 15 我也在做plasma membrane protein的phosphorylation,不过我是植物蛋白,加triton
x-
100或者sds让membrane protein soluble,然后centrifugation 取上清,sonication
是打
碎细胞膜吧,对膜蛋白不好吧。ELISA你用的什么抗体?我估计要用phosphotyrisine
threonine的
antibodies·做Western |
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o****e 发帖数: 1011 | 16 顺便问一下,那些彩色的,“粘在micro-centrifuge tube顶部的”label从哪里能买到? |
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C*****h 发帖数: 926 | 17 filter之后,在benchtop centrifuge里,离心10秒钟,去处少量debris
cells |
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s****a 发帖数: 454 | 18 updated experiments:
Experimental parameters:
(1) 15-50% (w/V) sucrose gradient, 10ml total, equal volume in each
fraction.
(2) Left two images: 15OD260 loaded, right two, 20OD loaded.
(3) Real time UV reading of fraction by Bio-Rad Biological LP, withdrawal
speed 3ml/min, except the top right, which was 2ml/min
Questions:
(1) How accurate are my marks of the ribosome complexes? I am not sure
which is 80S.
(2) Is my fractionation reliable and repeatable?
(3) It is after centrif... 阅读全帖 |
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a****d 发帖数: 1919 | 19 My experience:
heating up the elution buffer;
after mixing the eluted product with isopropanol,store at -20 for couple
hours,or overnight,then centrifuge. |
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n***w 发帖数: 2405 | 20 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 ... 阅读全帖 |
|
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s*****g 发帖数: 7857 | 22 在细胞量大而死细胞多时用Ficoll centrifuge分离.得到悬浮的活细胞.
而量少时就要耐心等待.慢慢长多了,再传代.否则有限的活细胞会丢失很多. |
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b*****e 发帖数: 288 | 23 至于用什么工具,
一看目的,
如果将来打算投各种biology方面的刊物,那只要选几个基因,搞个hypothesis,之后
的重点在拿出关键的生化、遗传方面的证据来。
如果是投各类omics方面的,那就得考虑一些大的范围、统计学、分子进化方面的结果。
如果投bioinformats方面的,得考虑算法以及可靠性的评估。
如果投system biology方面的,还得多方面构建Topology,最好能看见dynamic。
二看行业,
R在一些行业是基本工具。
如果将来打算专业做bioinformatcs的人不会用R,那不跟做生化的不会用centrifuger
一个道理嘛。 |
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n***g 发帖数: 5027 | 24 我就是查查资料,看看表达情况就可以了,并不想转行做生物信息了啊,呵呵
果。
centrifuger |
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s******y 发帖数: 28562 | 25 Native gel and sucrose gradient centrifugation |
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a*****y 发帖数: 269 | 27 spead should not > 4000xg |
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o*****r 发帖数: 156 | 28 This should not happen.
You know that you should never trust the Abs value when it's more than 2.
1. What is your reference in UV measurement?
2. Do you have anything in the buffer that absorbs at 280nm, for example
imidazole?
3. What't the extension coefficient of your protein?
5 ( |
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m**z 发帖数: 787 | 29 re this.What is the 280/260 ratio?
i had measured flow through from amicon before and it gave me high 280nm
reading. But when ran on a gel, there is nothing.
I think usually if you don't spin it with higher speed than you supposed to,
it should work just fine. |
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o*****r 发帖数: 156 | 30 Yes, spin at the recommended speed.
We use Amicon very often to concentrate proteins to ~10mg/ml.
I always put blank buffer in it and spin 10-15 mins, then spin my real
sample.
By doing this, the trace amount of glycerol (and other soluble stuff) on the
membrane get washed away.
And you do get a better UV measurement. My flow-thru was never higher than A
0.05 at 280 nm.
Occasionally, the protein might absorb to the membrane, but never ends in
the flow-thru more than 5%.
to, |
|
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s*******e 发帖数: 1010 | 32 what is in ur protein sample is most important. If it is hi-imidazole
elution, there will lots of small molecules have absorbance at 280nm and
they will go through membrane very easy |
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s**u 发帖数: 9035 | 33 All reagents, buffer need thaw and centrifuge throughly. |
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D*a 发帖数: 6830 | 34 Quick Tail Prep
Collect mice figure/tail in 0.5 ml Eppendorf tube. (4 mm is enough)
Add 100 microliter of quick tail digestion buffer (below).
Incubate at 55 C for 2 hours to overnight.
Vortex and incubate at 95 C for 10 minutes.
Centrifuge for 5 minutes to get rid of the small pieces of undigested sample.
(NOTE: I do neither vortex nor centifuge, and my pcr works
but if you vortex, you have to centifuge)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever ... 阅读全帖 |
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W*********n 发帖数: 775 | 35 please help me to have a look at the whole protocols and give some
suggesitons:
1) get 50midguts (about 3000cells/midgut, so about 1,500,000 cells or 80ug
protein totally),add 80ul lysis buffer (8M Urea, in 1XPBS, pH=8) including
Protease inhibitor and phosphatase, ultrasonic for 15-30 sec, then votex for
1 min. centrifuge for 10min at 14,000g and take the supernatant, add 16ul
5X loading buffer and heat for 20--30 min at 60 degree. load gel at 30ul/
lane and run for 2cm(for each sample, run th... 阅读全帖 |
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h******y 发帖数: 351 | 36 这家公司也在卖水稻表达的人血浆白蛋白。
http://www.amsbio.com/Animal-free-Human-Serum-Albumin-HSA-ecoHS
上几张他们网页上的图给大家看看,行家说说这样的活性算不算好。
Purity and Size of Competitive HSA Products by SDS-PAGE
Figure 1 below shows a Coomassie blue stained gel (left) and a silver
nitrate stained gel (right) comparing size and content of competitive HSA
products. The figure shows that there is no difference in migration patterns
or purity between: 1) ecoHSA™ , 2) a commercial human plasma serum
albumin purchased as a lyophilized ... 阅读全帖 |
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l*h 发帖数: 4124 | 37 If you calculate the centrifugal force (in g) and know the sedimentation
rate of your virion, you can have an educated guess about the conditions you
need.
the
). |
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m******g 发帖数: 69 | 38 Biochem Biophys Res Commun. 2010 Aug 6;398(4):723-9. Epub 2010 Jul 17.
Adipocyte-derived microvesicles contain RNA that is transported into
macrophages and might be secreted into blood circulation.
http://www.ncbi.nlm.nih.gov/pubmed/20621060
这篇paper说脂肪细胞分泌的microvesicle里面的RNA可以转移到macrophages里面,然
后macrophage的adiponeticn和resistin会升高。 他们的方法是,建立一个adipocyte
系3T3-L1,然后培养,然后把adipocyte的culture centrifuge, 提取出supernatant,
然后把supernatant进行ultracentrifuge,沉淀出这种microvesicle
请问这种方法是否可行,因为这步的关键是分离提纯microvesicl... 阅读全帖 |
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c********b 发帖数: 363 | 39 楼上的思路不错。
其实方法很多,但是比较简单的还是先crosslink,然后再用 harsh buffer把核裂开,
然后复性,最后该干嘛干嘛。
这里有一篇你可以参考:Rapid and phosphoinositol-dependent binding of the SWI
/SNF-like BAF complex to chromatin after T lymphocyte receptor signaling
我自己用的protocol是这样的:
如果只是检测蛋白表达:
用High SDS buffer:
15 mM Tris pH 8.8, 5% SDS
0.3 M DTT, 15% Glycerol
我的是植物细胞,一般是liquid nitrogen grind之后加buffer,boil.如果是cell line,
collect细胞之后加上buffer 然后直接煮.除非是非常不稳定的蛋白,不然连protease
inhibitor都可以不加.
看到蛋白之后如果是做IP:
先crosslonk(用什么chemical得自己琢磨),然后把ripa的SDS含量加到1% ,... 阅读全帖 |
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y******8 发帖数: 1764 | 40 Label after fix is easier for you. e.g. antibody.
However, if the resolution is not high enough, I doubt that you will get any
better result than ultra-centrifugation fractionation. |
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p*l 发帖数: 1359 | 41 自己必须有incubator,bio-safety hood, centrifuge。 water bath,pipette,
balance, ph meter 这些你们化学实验多半也是有的。-80冰箱,液氮罐用得不多的话
可以蹭蹭别人的,我就是一直在蹭。关键是得有个小屋关起门来专门来做cell culture
room。 |
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m******5 发帖数: 1383 | 42 ...... I am very uncomfortable to say that we have neither a reliable
centrifuge nor fraction collection machine nearby... |
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s****r 发帖数: 736 | 43 老企业,多用板框,但是清洗和收集得劳动强度太大。
现在新型药厂或者生物制剂公司多用continuous centrifuge或者membrane (e.g.
ceramic ) filtration. |
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l**********1 发帖数: 5204 | 44 Please go to
//www.ncbi.nlm.nih.gov/pmc/articles/PMC2518226/
>Overall, our results suggest that PUB22 and PUB23 coordinately control a
drought signaling pathway by ubiquitinating cytosolic RPN12a in Arabidopsis.
Subcellular Localization
The sGFP cDNA clone was fused in-frame to the 59 end of the full-length
PUB22 and PUB23 coding region and ligated into the pBI221 transient
expression vector. The sGFP-PUB22 and sGFP-PUB23 fusion constructs
were transformed into protoplasts prepared from wild-typ... 阅读全帖 |
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D*a 发帖数: 6830 | 45 Quick Tail Prep
Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
Eppendorf tube.
Add 100 microliter of quick tail digestion buffer (below).
Incubate @ 56 C several hours to overnight.
Vortex and incubate @ 99 C, 10 minutes.
Centrifuge @ max speed, 5 minutes.
(NB i don't vortex so i don't need to centifuge the tube, it works)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever you routinely do).
Transfer supernatant to a fresh tube for s... 阅读全帖 |
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D*a 发帖数: 6830 | 46 Quick Tail Prep
Collect 1 mg skin biopsy (= normal size tail or figure tip) in 0.5 ml
Eppendorf tube.
Add 100 microliter of quick tail digestion buffer (below).
Incubate @ 56 C several hours to overnight.
Vortex and incubate @ 99 C, 10 minutes.
Centrifuge @ max speed, 5 minutes.
(NB i don't vortex so i don't need to centifuge the tube, it works)
Use 1-2 microliter aliquot of supernatant for a 25-50 microliter PCR
reaction (or whatever you routinely do).
Transfer supernatant to a fresh tube for s... 阅读全帖 |
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b******y 发帖数: 627 | 47 yes, there is well established sucrose gradient centrifugation method. |
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b*******n 发帖数: 605 | 49 要给试验室买一个bench centrifuge,就是可以转PCR plate, 50ml, 15 ml tube那种,
quote了eppendorf 5810R 和 beckman Allegra X-14R. 这两个型号应该是
compatative 的,但为什么5810R 比 X-14R 要贵2000 多呢?
现在钱不是问题,但是eppendorf的representative牛皮哄哄的,很不招我喜欢,问一
下哈,凭什么一定要买eppendorf呢?多花2000块,到底值不值? |
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h**********r 发帖数: 671 | 50 主要是plasmid,过柱子洗脱体积太小洗下来吧?
多谢! |
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