i*****g
发帖数: 11893 | 1 正好这几天干自己的事情，搜索 desalting的办法
看见了 pierce 有一堆产品
LZ可以串联两种方法，估计近乎100% desalting
http://www.thermo.com.cn/article.aspx?id=5157
Zeba desalting and sephadex，PAGE gel desalting
这些方法，无非两种，ion exchange, gel filtration，他们现成做好了产品可以用 |
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j****x
发帖数: 1704 | 2 普通PCR，引物长度在25以下的话desalted足够了，大部分qPCR用desalted也没什么问
题。长度超过30bp，特别是用于突变的，推荐用HPLC/PAGE纯化的，但是价格自然就飙
升十倍以上了。不过听很多人讲用desalted的引物做突变也没什么问题。
记得以前在国内的时候，合成引物的默认纯化方式就是PAGE，很便宜价格没这么离谱啊
，不知道为什么 |
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d*********e
发帖数: 15 | 3 I measured original solution without desalting too and uv 280 reading was
consistent with concentration after desalting. Just very very high uv260
reading. What would cause high uv 260 reading ? It is not nucleic acid.
After desalting, uv280 >uv260.(something like uv280 0.4, uv260 0.2.).
Did FDA come to measure protein concentration ? I doubt it.
FDA should measure protein concentration from samples randomly taking from
phamacy.
There are a lot of pharma dirty things exposed before, such as |
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m**z
发帖数: 787 | 4 dialysis不就行了，干吗非要desalting column呢
desalting |
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w******e
发帖数: 1187 | 5 RNA: in vitro transcription＋phenol＋Urea PAGE purification＋elutrap＋
column desalt。
radiolabeling：artarctic phosphatase，heat inactivate。PNK＋P32 ATP，
heat inactivate。column purify+desalt
thx! |
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发帖数: 1 | 6 Genome-editing
下午4:50（6 分钟前）
其他收件人： h********[email protected], c******[email protected], o*****[email protected]
将帖子翻译为中文
- 显示引用文字 -
hello guys,
we tried NgAgo plasmid cotransfection with 5'phospho-ssDNA guides and I must
say cell mortality rates are very high (toxic). We got 5'phospo-ssDNA
guides synthesised from IDT, desalt purification. Does anybody have tested
desalted verus PAGE purified ssOligos and looked for cell viability.
cheers! |
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w*********g
发帖数: 30882 | 7 Think about the conflict between the Theocracy & Evolution Theory, you will
understand the current situation. The point is too advanced tech will
greatly weaken capatalists' control over people, since energy, desalted
water and food will be free.
====================================================================
更高的角度看现在的形势
来源: 牛牛哥 于 2012-03-29 08:42:40 [档案] [博客] 旧帖] [转至博客] [给我悄悄
话] 本文已被阅读：119次 字体：调大/调小/重置 | 加入书签 | 打印 | 所有跟帖 |
加跟贴 | 查看当前最热讨论主题 人类已经到了一个临界点
1。科技
2009年科技突破了红光和蓝光的隐性技术，但是预计制造出神话中对自... 阅读全帖 |
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w*********g
发帖数: 30882 | 8
China has mature technique of desalt sea water by using membranes and rich
experiences in turning deserts into grasslands. |
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s****r
发帖数: 31686 | 9 no desaltation device on liao nin ship
us ship is nuk powered, too much energy produced
而且美国航母上通常有海水淡化装置，淡水自制，不需要补给。不知道辽宁号是不是够
先进，也安装了海水淡化。
★ Sent from iPhone App: iReader Mitbbs Lite 7.56 |
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R****a
发帖数: 6858 | 10 In my point of view, the following techs are promising
1, 3D printing.
2, nuclear fusion reactor.
3, quantum communication and quantum transistor
4, on chip optical interconnect
5, mass sea water desalting.
6, carbon fiber.
7, gene medicine and organ copy.
8, plasma engines.
9, gene improved plants
10, electricity drived car/floating autos |
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w********2
发帖数: 632 | 11 McKinley Conway, 1920-2011:
Last Flight Home in a Landmark Life
ONLINE INSIDER
McKinley Conway
by JACK LYNE
T
his Mac Conway fellow, you might be wondering, now just who was he again?
Yeah, he's the guy who started Site Selection 'n all, but did he ever do
anything else? I'm kinda in a hurry here, pal. So just tell me who this dude
was like.
Oh, hell, Mac Conway wasn't like anybody. He never followed anyone's
footsteps; he couldn't really. He was too restless a spirit, too hell-bent
on slashing ... 阅读全帖 |
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s**********8
发帖数: 25265 | 12 ph may be critical for isoelec point for protein. or if cation ion change
mechanism, low ph is required too. salt not too bad for mass spec. you can
use ziptip or similar product to desalt.
10. |
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G*********o
发帖数: 49669 | 13 吃了吗？
ph may be critical for isoelec point for protein. or if cation ion change
mechanism, low ph is required too. salt not too bad for mass spec. you can
use ziptip or similar product to desalt.
10. |
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r****o
发帖数: 105 | 14 I have similar experiences as yours. That is about determination
the anomericity of glycosylation on EGF repeats. It involves
processing the small domain EGF into peptides, and then subject them
to glucosidase digestion.
I would suggest you using HPLC with either Reverse Phase or Gel filtration
column(like superdex from Pharamacia). This is the best way to recover
small amount of peptides. If your sample has quite a lot and the MW is
big enough, you can try acetone precitation to get rid of |
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r****o
发帖数: 105 | 15 1 design primers of ~30 bp length.
2.Use good polymerase to do PCR (herculase from stratagene is great,
a Pfu with special buffer in it to increase the fidelity)
3. add 1~3 ul Dpn I directly to the PCR reaction tube. digest it
for at least 3 hours in 37 degree
4.Desalt the PCR sample with PCR purification kit from Qiagen.
5.Transform competent E.Coli.
6. Pick up several colonies , grow small cultures and do miniprep.
7. Send the DNA to sequencing. |
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r****o
发帖数: 105 | 16 1 design primers of ~30 bp length.
2.Use good polymerase to do PCR (herculase from stratagene is great,
a Pfu with special buffer in it to increase the fidelity)
3. add 1~3 ul Dpn I directly to the PCR reaction tube. digest it
for at least 3 hours in 37 degree
4.Desalt the PCR sample with PCR purification kit from Qiagen.
5.Transform competent E.Coli.
6. Pick up several colonies , grow small cultures and do miniprep.
7. Send the DNA to sequencing. |
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f*********p
发帖数: 13 | 17 desalting column is much faster, but sometimes proteins bind to it
non-specifically and you may lose some of your sample by that. sometimes,
proteins get denatured because the buffer change is too sudden, not like
dialysis that is slow and "tender". plus, carry over is almost inevitable on
the column, and a 2nd run is sometimes necessary.
dialysis on the other hand, has its disadvantage too. most of all, it takes
way too long (O/N is routine), during which the protein may not be happy.
second, i |
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l****y
发帖数: 398 | 18 desalt, dry and weigh
换算 |
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C*******e
发帖数: 4348 | 19 恩,总的来说IDT还是比别的好
不过有些extra purification steps是不是那么好就不好说了,
我又一次需要P32标记primer,订的是HPLC纯化的,
可是标记下来以后发现本身primer就不纯,
应该只有一个大小的,其实有三种不同大小
后来需要标记的primer我就订最普通的desalt的
自己PAGE胶纯化切下来回收
当然如果不是P32标记可能也看不出来其实有三条不同大小的带 |
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T**********t
发帖数: 1604 | 20 有多便宜啊，我想上去看看价格比较一下，还不给我看，要request ordering access
先，够麻烦的。
能不能麻烦谁帮我看看随便一个20bp的RNA片段，1 umole scale，desalt的价格在
sigma是多少？ |
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T**********t
发帖数: 1604 | 21 用desalting column试试呢？
或者你把已经有沉淀的样品离心一下，取上清再加点SDS再跑胶。如果你之前加的
loading buffer里的SDS已经把K离子都沉淀下来了的话，应该后来加的SDS就不会沉淀了吧。
不过我不知道你的蛋白是不是也跟SDS一起沉淀了，还是先除盐比较保险。 |
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M*******a
发帖数: 15 | 22 concentrate & desalt culture supernant --> proteomics
If you have target proteins in mind --> immunoblot |
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c******r
发帖数: 3778 | 23 嗯，可能原因比较多。
1. 不知道你的primer有多长？有没有HPLC purify？如果cloning可能仅仅desalt是不
够的，尤其是比较长的primer。但是5'少掉比较奇怪，一般合成都是3'少掉几个？
2. 不知道你用的什么polymerase？会不会有5’exonuclease activity？
要不你试试随便加4个base pairs再做一下，就算再少4个base pairs也没关系了。 |
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H*g
发帖数: 2333 | 24 准备用纯化的his-tag蛋白做EMSA，不知道imidazole会不会对EMSA有很大影响。
请教大家，需要事先脱盐除掉imidazole(300mM)吗？不知道要不要老板去买desalting
的柱子。
Thanks！ |
|
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I*****y
发帖数: 6402 | 26 在我以前用过的系统里面，300 mM imidazole影响EMSA的binding, oligomerization
desalting |
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H*g
发帖数: 2333 | 27 Thanks for all the feedbacks!
Does Dialysis take too much time? I am just too lazy, since desalting column
seems easier. |
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T**********t
发帖数: 1604 | 28 我在ITC订的oligos一般即使只是desalted的纯度也都很好，我跑HPLC看过。 |
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m**********2
发帖数: 6568 | 29 labor cost. PAGE can't be fully automated. Desalting is. |
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C*******e
发帖数: 4348 | 30 理论上说应该没有问题
不过可能要摸条件
我用70-80，还有>100的primer做过regular PCR
不过我用的primer
1.Fwd和Rev差不多等长，Tm接近，
而且priming sequence (for target gene)部分的Tm也接近
2.我用的priming sequence比较长，24-28nts
我觉得你做的时候可能
1.不仅reaction的条件要摸
2.PCR cycle的设定要摸（除了gradient，还有touch down，touch up，3 steps, 2
steps之类的可以试）
3.有可能template也要试,我遇到过用整个plasmid不行，
把target gene的fragment切下来胶纯化以后做模板就没有问题
4.如果一切不顺利，建议reverse primer也设计个长点的
哦对了，还有一点，长的那个primer，最好要订纯化过的
不用HPLC纯化的也要PAGE纯化的
或者订了std desalted的回来自己用PAGE胶纯化一下
越长的primer越有可能5'段少一些（对于>100 nts的primer可能少的是一... 阅读全帖 |
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K******S
发帖数: 10109 | 31 简单的说，DYNAMIC RANGE就是说样品里浓度的差别。
拿SERUM来说吧，ALBU的浓度和IL-6浓度差10个数量级。LCMSMS有LOD和饱和的问题，一
般DYNAMIC RANGE是3-4个数量级，所以没有很强的前期的SEPARATION的话，你能检测到
得都是含量最高的那些蛋白。
如果是我的话，我一开始就做CELL LYSIS, IN SOLUTION DIGESTION, DESALTING, 然后
直接做LCMSMS （让他们RUN一个长的LC GRADIENT），一般也能出来几百个蛋白，先看
看你的样品里有什么东西，再设计下面的实验。这样你一开始是要付4个样品的钱。 |
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x*****o
发帖数: 441 | 32 DNA 还是RNA? 买来是deprotected的吗?
要是已经deprotected的了,就是你水加得不够,多加点水应该就溶了. 胶smear有可能
是orverload 或者盐分太多. 如果是overload就用大点的 well, 如果是盐就desalt一
下,或者乙醇沉淀一下或者过个柱子.
。。 |
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p******i
发帖数: 1092 | 33 虽然SC说抗体能IP，建议你最好用自己的sample做个routine的IP+Western试一下。确
定能IP了，再考虑CROSS LINK？
有关重复用agarose beads的问题
1 IP需要多少抗体？没觉得省很多啊……既然用的是原代细胞，你的DISSECTION和
CULTURE的人工费用应该比抗体贵很多吧
2 要确保经过 1和DSS反应 2被ELUTION buffer摧残 后的抗体仍然好用……
浓缩洗脱组分（随便换buffer或者desalt）可以用spin column，
参见
http://www.life.sci.qut.edu.au/epping/LSB607OLT/607concentrator |
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i*S
发帖数: 175 | 34 我用的Pierce卖的梯度胶，然后sypro ruby染色。图是用机器扫出来的。用肉眼在
transilluminator上看也差不多。
能看见条带，可是lane里面背景太高，很不分明。特别是边缘像是smear了。请问大家
有没有什么主意？
我开始认为跑之前我没desalting，溶液里Na+ 和 Ka+ 大概都约500mM，有些高了，蛋
白分离的不好，于是用氯仿提纯了一下，好像没什么帮助啊……
PS: 下方黑块是怎么回事我也不清楚嗯 |
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s******y
发帖数: 28562 | 35 How big is your protein? If bigger than 30kD you can use
mini desalting column with sephadex G-25. Done in less than 5 minutes.
If smaller than 30kD you can use sephadex G-10, or smaller. |
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b******y
发帖数: 627 | 36 For LZ,
sunnyday's desalting method is the best to try. I will be surprised if it
doesn't work. I guess you have to use a syringe because nobody likes to
inject 32P into their FPLC. In that case, you should be careful of the
fraction size. Don't mix two well separated peaks during fractionation.
kaka1000's vivapure spin column离子交换柱 is a cation/anion exchange column,
to which unlimited volume can be loaded. In the end, elute with B buffer.
The peak is sharp because the salt concentration is high ... 阅读全帖 |
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i*****g
发帖数: 11893 | 37 楼主为什么要上HPLC？ facility people will not be happy about the P32.
G10, G25 may be a good choice to desalt the ATP, and it might lose some
proteins.
centricom or centricom-like spin column is suitable for >10ug protein. ug
amount of proteins from the phosphorylation assay could be lost after spin
column concentration. |
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N2
发帖数: 81 | 38 一般的快速脱盐的柱子不能够达到你100%脱盐的要求.因为为保证回收录,洗的体积大,
总是有残留.
我们以前常用GE 的Disposable PD-10 Desalting Columns. 大约脱掉90%的样子.如果
这种柱子短,其实自己做一个长的就行了.重力洗脱就足够快了.
我建议楼主要有一个好的流程和组份的控制.
1)做一个预实验,如果有足够的样品.把洗脱液分成100-200ul收集. 然后做 Liquid
scintillation counting 32P,这样可知最大可收集的,没有32P的洗脱体积.
2)如果不希望太稀释样品,最前面的洗脱组分,用nanodrop测280nm吸收,没有多少蛋白的
,不要就好. |
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s******9
发帖数: 283 | 39 透析或过desalting column，蛋白提纯的常规操作。 |
|
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b***n
发帖数: 36 | 41 哥们，我有遇到过跟你类似的问题，蛋白跑着跑着就跑没了
后来发现是提出来的蛋白本身就不大稳定，容易形成寡聚（跑ＨＰＬＣ可以看出问题）
很大的一部分直接就聚集在Ｓ２００的上端的ｆｉｌｔｅｒ上了。
提高点盐浓度有些帮助，不过帮助不大
你的实验目的是啥呢？
我的主要是为了除盐纯化，所以后来改用５ＭＬ的Hitrap Desalting Column来跑，
基本上就不损失了
很能理解好不容易用昆虫细胞提到点蛋白跑着跑着就跑没了的哀伤，，，，，
然后，请教了做结晶的童鞋，基本上都是说这种事情是由蛋品决定的，，，如果不是非
玩不可的话，能不能换一个蛋白呢，，， |
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g*********5
发帖数: 2533 | 42 rna 蛋白相互作用
想直接从公司 order rna 5‘ 标记的oligo, 50 bp long。 IDT could link Licor
dye to the RNA.So use it I could see the shift directly from gel.
大家order rna的时候，是不是 都要用hplc纯化？
charge another 75刀
脱盐不要钱, but I don't know if the desalt quality is fine for the RNA-shift
experiment.
thanks |
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i*********0
发帖数: 915 | 43 cre + beta-actin 的primer，定了好几个batch（MWG)，基本都是spear的带。
今天从隔壁实验室拿了一点他们的cre 和beta-actin的primer，是metabion的，同样的
浓度，同样的enzyme，结果出来很好。
懂行的说一下，效果为什么差这么大？
这些primer都是那种desalt的。 |
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z*********3
发帖数: 335 | 44 I used to order from Sigma (100 to 150 bp), PAGE purified grade, and ask
Sigma to provide the PAGE gel picture for show the quality of the oligo.
Normally, <60 bp is purified by desalt column. HPLC comes between 60 to 100.
PAGE is the best for >100 bp. IMO. |
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v**********t
发帖数: 9 | 45 从Dharmacon订的RNA，25mer, single-strand, HPLC purified, deprotected,
desalted.
拿到手后用DEPC water resuspend, 加到蛋白溶液后就把蛋白给沉淀了。先后订的三
批RNA全这样。
哪位知道这是怎么回事？ |
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C******T
发帖数: 300 | 46 并不是所有公司会给出peptide content。据我的经验，纯度数据会免费提供。大部分
公司peptide content会额外收费，一般100左右，贵的可能会200到300。我同意你的说
法，LCMS可以给杂质定性。有两点我觉得可以商榷，第一，透析能不能去除盐。我们一
般先desalt, 然后HPLC提纯多肽（～95% pure)，然后做peptide content(假设70%)。
这25%的difference一般是多肽上结合的盐，比如TFA。众所周知多肽是以盐的结合体形
式提纯出来的。透析是不必要也是无益的。第二，多肽的不纯物当然会影响到结果。关
于小分子对生物学有没有影响，别的杂质不说，就举一个简单的TFA。业界的animal
study protocol，一般会要求避免多肽以TFA盐的形式进入动物体内，首选是acetate
salt，病理学毒性比TFA小 up to 30%。其他的小分子就更不用提了。TFA影响生物学的
例子我自己也见过一些。 |
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p**********m
发帖数: 472 | 47 这不就是desalt的吗， 我觉的用C18 cartridge 足够了 |
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M*****9
发帖数: 320 | 48 For desalting, PD10 also works. |
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h*****t
发帖数: 1226 | 49 NanoSpray Tip不稳定和堵塞是常见问题。
注意Tip size， 溶剂的过滤和样品的desalting |
|
c******n
发帖数: 16403 | 50 你们搞这么复杂做什么， 拿个desalt cartridge就搞定了。 本质就是个反相柱。
合成DNA或者peptide的实验室基本都有这些东西
酸。
KCl |
|
