N*****r
发帖数: 2108 | 1 这个我也没找到什么合适的纸。
国内的朋友很多人用手揉纸（通常用来包鲜花的）、薄牛皮（40g）还有蜡纸（非常薄
，好像不是我们用来包食物的那种），不过我在美国没找到类似的东西。
我是用的tissue paper，两张加胶水（craft glue[water soluble]）粘在一起。 |
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c*****t
发帖数: 1879 | 2 The solubility of protein depend on several factors, listed
in term of importance.
1. The protein itself.
2. pH.
3. Temperature.
4. Salt.
5. Buffer. |
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r****o
发帖数: 105 | 3 Do you have a way to distinguish correctly folded protein? If you have,
just use HPLC to purify them from crude E.Coli expressed protein.
If you don't have one way to do that.
Refolding involves (1) denaturing and reducing (2) suddently dilute
the sample extensively to mild condition (3) get the soluble protein.
Try to search pubmed for methods or refer to any classic protein biochemistry
protocol book. Note: the yield is low(less than 10% of the crude protein
will be correctly refolded). |
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m**o
发帖数: 13 | 4 I am not so sure if RNase keep active in such a solution containing 50% or so
isopropanol. However, For purification purpose, it is not suggested to have a
nuclear acid precipitated with isopropanol method either under low temperature
or for long incubation. Salts often have lower solubility in isopropanol
solution than in ethanol solution. |
|
n****e
发帖数: 1677 | 5 I used to make different constructs for 1 protein, they are all soluble, but
behave different, some are monomer, inactive, some are dimer inactive, some
are dimer active can't crystallize, some are dimer active can
crystallize......
And the difference between these constructs are just around 10 residues.
所不
另一
而不
来就 |
|
a******u
发帖数: 211 | 6 1. Therapeutic vaccination in patients with gastrointestinal malignancies. A
review of immunological and clinical results.
Mosolits S, Ullenhag G, Mellstedt H.
Ann Oncol. 2005 Jun;16(6):847-62. Epub 2005 Apr 13.
2.Colorectal cancer vaccines: what we know and what we don't yet know.
von Mehren M.
Surg Oncol Clin N Am. 2007 Oct;16(4):873-900
3.Soluble tumor-associated antigens in cancer detection, prevention and
therapy.
Zusman I.
Med Sci Monit. 2004 Dec;10(12):RA317-24
请发paper到e*********[email protected] |
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a***e
发帖数: 1010 | 7 yeah, you need refolding most of time.
An alternative method is adding some sarkosyl during purification, which
solublizes your protein into the supernatant. |
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m****n
发帖数: 37 | 8 Abeta plaques was considered to be the cause of AD a decade ago. Now, people
think soluble Abeta is more likely to be the cause, while Abeta plaques are
just the by product.
Genetic studies provide strong evidence that APP pathway play the pivot
role in AD. Tau also has its share in the game.
However, for sporadic AD patients, no solid evidence yet for any hypothesis. |
|
p****s
发帖数: 3153 | 9 I am doing something related to it, not the AD itself
The popular view is that the soluble oligomers of Abeta is the main cause of
the disease, proposed by big bull Chris Dobson and others. Anyway, Abeta is
a great model for biophysicists, so if it really causes the disease is a
less important questions for them, at least for me. XD |
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q********n
发帖数: 321 | 10 Most multiple plates are made of PC or PS. They will be damaged by
chloroform. Search for PP or PE plate or check polymer solublity in DMSO. |
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P******r
发帖数: 11 | 11 有人很懂ATPs (aquae two phases system)的请帮忙.
Whether it will work for removal of all soluble proteins (bulk proteins)
from protein mixture in HEPES solution?
Condition: PEG1450 (low molecule weight); High pH up to 12; 1M (NH4)2SO4; 0.
5M NaCl. |
|
v***a
发帖数: 1242 | 12 2篇paper，看到 Informa 有，但是下不了。。。
A Distinct mRNA Encoding a Soluble Form of ICAM-1 Molecule Expressed in
Human Tissues
Cell Adhes Commun. 1995 Nov;3(4):283-92.
Monoclonal Antibody-Conjugated Immunotherapy of Cancer
Int Rev Immunol. 1997;14(2-3):213-27.
email：
v****[email protected]
多谢！！ |
|
|
p*****m
发帖数: 7030 | 14 话说HD现在大家也都认为soluble htt而不是htt包涵体才是致病原因（有些年头了）。
不知道AD的人是不是潜意识里受了HD的进展的影响 lol 或者过几年大家就发现所有的
蛋白变性疾病 变性以后的玩意都是保护机制。。
dem
很可
保护
发展 |
|
s******y
发帖数: 28562 | 15 Depnding on really what do you need to do.
If you want to simply seperate the agregate from soluble protein,
you can buy those 10 cm disposable columns and pack the resin by yourself.
It is not too difficult.
Or, you can just do a ultracentrifugation with a sucrose cushion to seperate
the aggregate. |
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a****k
发帖数: 1130 | 16 don't let the RNA pellet dry completely, it'll greatly decrease its
solubility
I
Thanks |
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T**********t
发帖数: 1604 | 17 主要是让蛋白变性沉积，跟covalent bond没啥关系。
http://en.wikipedia.org/wiki/Fixation_%28histology%29
Precipitating fixatives - Alcohols
Precipitating (or denaturing) fixatives act by reducing the solubility of
protein molecules and (often) by disrupting the hydrophobic interactions
that give many proteins their tertiary structure. The precipitation and
aggregation of proteins is a very different process from the crosslinking
that occurs with the aldehyde fixatives.
The most common precipitating fixatives are et... 阅读全帖 |
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s********n
发帖数: 2939 | 18 从current protocol上改的：
1. check OD600；
2. harvest X ul induced or uninduced culture;
3. add Y ul 1x SDS-PAGE loading buffer (Y=OD600*X/10);
4. Vortex for 30s;
5. 100C for 5 min;
6. 15,000x g for 5 min;
7. Load 5-20 ul supernatant to SDS-PAGE gel.
你也可以向楼上所说，把soluble和insoluble的fractions分离后再SDS-PAGE. |
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U*****t
发帖数: 19 | 19 参考了楼上的答案后给出我回答
酶切+质谱，质谱-质谱应该能确证该蛋白是否存在目标蛋白的片段。
首选 Western Blot；
辅助证明 酶切，看是否产生目标蛋白酶切的片段。
蛋白与它的目标蛋白反应--------- 如用 SPR， Fotebio 等不需纯化好的蛋白
CD， DSC, temperature-dependent fluorescence (有不同称呼 如 Tdf 或
thermFluor）
N-端 去 Met；磷酸化，。。。
质谱-质谱可以 mapping 哪儿进行了修饰
SDS-PAGE 后 Glyco-staining
solubility screening 获得好的溶解条件。一个一个条件地试是比较傻的。 |
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m**z
发帖数: 787 | 20 run gel on the following samples: -IPTG, +IPTG, lysis supernatant, lysis
pellet.
If you have protein in +IPTG sample, then if it is mostly in pellet, that
tells you the solubility in this buffer is probably not great. |
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A****w
发帖数: 244 | 21 跑个胶，拿出杂质蛋白，做个trypsin digested MALDI看看杂质是什么。
50%的soluble是不是分离的蛋白可能是聚集体？试试低温表达。
有loop不稳定结构，加arg+glu到buffer里，是不是可以增加蛋白的溶解特性。
暂时就想到这些。 |
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c******r
发帖数: 3778 | 22 实验嘛，不行就自己发明啦。
比如可以先回答cell contact是否required for somatic cell induced stem cell
proliferation.
可以把somatic cell先做你想做的处理，然后放在culture里，跟stem cell用0.3um的
膜分开。这样如果stem cell proliferation增加了，那说明是soluble factor，在
culture medium里面。可以尝试用不同的antibody block。这样可以找到是什么factor。
如果stem cell不增加，那么可以把stem cell用CFSE之类的marker标记了，和处理过的
somatic cell混养，这样如果刺激stem cell proliferation，那么说明cell contact
很重要。那么可以focus on cell surface molecules，试试不同的knock down，看看
有没有哪个能block这个过程。
当然，注意做好controls。
具体实验设计要看情况，我的意思是你这个问题很有意思，不能因为... 阅读全帖 |
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r******y
发帖数: 21907 | 23 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 |
|
p********i
发帖数: 116 | 24 最近发现一个蛋白A，可以另外一个蛋白B binding并且影响B的soluble和insoluble的
分布，但是B的总量
不变，感觉A是不是chaperone, 但是做染色和ER的marker没有在一起定位，这就能说明
这个不是个
chaperone了吗？ |
|
m********r
发帖数: 124 | 25 我似乎前面已经发过帖子了，但是过了两周后，对这个问题知道的更多了，也有新
的疑惑，特来请教。
我的蛋白13KDa在连了N-端GB1tag后solubility增加了，也能拿到纯的，但是切掉
tag后就找不到我的蛋白了。我们一般用Ni柱，tag挂在柱上（因为有6xhis），目的蛋
白在flow-thorough.但是这个FL跑胶没东西。不是量的问题，因为浓缩后也没条带。（
我试过GST-tag，类似的结果，而且不加tag的时候我基本看不到表达条带）现在我怀疑
是降解掉了，因为上个帖子说过，结构预测N-terminal没结构，之后C-terminal 70aa
有一些二级结构。问题是在切掉tag前他已经很纯了，为什么切了tag还会降解呢？是不
是因为本身不稳定，跟有没有protease没关系？还有既然如此，我以后的生化试验是不
只能连着tag做了？唉，希望这个tag不会影响吧。 |
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n***3
发帖数: 663 | 26 1.J Control Release. 1999 Aug 27;61(1-2):233-40.
Positively charged liposome functions as an efficient immunoadjuvant in
inducing cell-mediated immune response to soluble proteins.
Nakanishi T, Kunisawa J, Hayashi A, Tsutsumi Y, Kubo K, Nakagawa S,
Nakanishi M, Tanaka K, Mayumi T.
2.J Control Release. 2008 Aug 25;130(1):22-8. Epub 2008 May 15.
Reactive oxygen species play a central role in the activity of cationic
liposome based cancer vaccine.
Yan W, Chen W, Huang L.
Please send the paper to em... 阅读全帖 |
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e*****y
发帖数: 146 | 27 最近要用一种刺激剂，加到细胞里去的，说明书里说是The product is soluble in
ethanol，insoluble in water.
于是不知道应该用什么乙醇配制了，实验室里的都是些外用的分析纯乙醇，有95%的，
100%的，好像没有用来配制细胞试验试剂的“无菌”乙醇，大家有过类似的经验吗？我
能用这些100%的分析纯乙醇来配制试剂吗？会不会导致细胞污染呢？...... |
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z*******6
发帖数: 679 | 28 I don't think the selection marker matters a lot. I think it is the cell
line and the way of transfection that matters more. If you don't care the
expression level tooooo much, lentivirus works really good. But last time I
was doing A549 and I need the expression level to be really high to secrete
a lot of soluble proteins for ELISA detection, none of methods (
electroporation, nucleous transfection, lentivirus) or selection markers (I
tried multiple ones) worked. |
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h********r
发帖数: 519 | 29 现在最重点的是soluble ab oligomers, rather than ab fibrils or monomers, are the most toxic species. The origin of ab toxicity may come from the membrane-disruption. Ca overload may be consequence of membrane disruption due to the formation of ion channel. |
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h*******o
发帖数: 4884 | 30 1) due to the intrinsic difference between rodent Abeta and human Abeta,
rodent Abeta do not form aggregates easily as human abeta.
For more detailed info, check "Charles G. Glabe" or Frank Laferla's paper.
As for NFT, most people in the field now agree that Tau is downstream of
Abeta.
2) Calcium and neurocytotoxicity hypothesis is quite old. It definitely has
its points but not the whole scheme.
3) Abeta is solube. The problem is Abeta oligomer. And the current opinion
is that soluble Abeta ol... 阅读全帖 |
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D*****l
发帖数: 554 | 31 We have developed protocols to obtain keratinocytes, the primary cell type
found in the epidermis, from hESCs by the proper temporal addition of
extracellular matrix, growth factors, cytokines, and other soluble chemical
agents. Our current efforts focus on characterizing the function of these
hESC-derived keratinocytes in wound healing applications, and generating
other types of skin cells from hESCs.
http://paleceklab.che.wisc.edu/hescs_skin.htm |
|
g*********r
发帖数: 281 | 32 Maybe it is not necessary to ask, where did you put your GST? Normally N-
terminal GST will help your protein folding and solubility.
In term of inclusion body, like people mentioned, lower temperature and IPTG
concentration might help.
Or try some other Tag, like MBP. |
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w*********e
发帖数: 98 | 33 Try low temperature at 18 C, which should start right after you inoculate
the overnight culture into the final culture. Then wait until OD is 0.6-1.0,
add low concentration of IPTG such as 0.1 mM IPTG for 16-20 hours at 300rpm
. I just tried this protocol, it increased a lot the ratio of GST fused
protein in soluble fraction compared with that at 37 C. |
|
n***w
发帖数: 2405 | 34 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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w*********e
发帖数: 98 | 35 I used 30ml because I was trying to see if my protein could be expressed.
After IPTG was added, the culture was shaked at 18 C for 20 hours in order
to get enough cells.
The big difference based on your protocol is that I started low temperature
the next morning when I did final inoculation, while you still used 37 C.
I compared the soluble and the pellet fractions at 18, 28 and 37 C,so I know
18 C is the best one. |
|
x*******i
发帖数: 32 | 36 Thanks! But why the philic environment causes emission red shift?
Bascially we have a protein soluble at room temperature but it precipitates
at cold temperature such as 4 degree. It re-dissolves when temperature
increases. I am thinking about fluorescence spectroscopy to see compactness
or openness of protein with temperature. |
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z*h
发帖数: 773 | 37 The simplest explanation is solubility vs temperature. |
|
e****s
发帖数: 1125 | 38 How about sequence similarity between these domains?
Elisa, all domains are in soluble conformation. A's small region could
be folded inside.
In WB, all domains and proteins are denatured. If there are some highly
conserved region between B,C, and D, then I think you got an explanation.
A.
Full |
|
x*******i
发帖数: 32 | 39 Hello There,
We have a protein (pI = 8.9, 10 mg/mL, MW 100 K) precipitated in 10 mM
phosphate buffer (pH around 7) after overnight storage at 4 degree. The
precipitate will be soluble again when warming up solution to room
temperature. Addition of NaCl will prevent precipitation. Other common
buffers are OK not causing precipitate.
Hypothesis is ionic cross-linking. Phosphate ion has intense negative charge
density and working as the cross-linking agent, its multiple negative
charges interact wi... 阅读全帖 |
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s********n
发帖数: 2939 | 40 我建议你还可以保留一部分样品，做supernatant和precipitation的比较，可以看出是
否是soluble expression。 |
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h******y
发帖数: 351 | 41 What kind of cells, which fatty acids are you using? Please give more
details.
Here is what I used for hepatocyte.
Preparation of the fatty acid stocks
The free fatty acids are prepared by dissolving each individual free fatty
acid in 100% ethanol. Most are readily dissolved in ethanol, though some
require heating to achieve fully solubility.
A final concentration of 100 mM for each of the fatty acids as follows:
31 ul 1 M Palmitic Acid Solid, 1 g / 3.3 ml EtOH
... 阅读全帖 |
|
n***w
发帖数: 2405 | 42 Because most of my protein is in inclusion bodies and I don't wanna directly
go to the last resort dissolving
with guanidine hydrochloride, I found a protocol using this detergent to
increase the production of soluble
protein.
I know nothing about its physical chemical properties and I haven't thought
about the impact of this
ingredient.
Thanks. |
|
C*******e
发帖数: 4348 | 43 soluble aggregation是啥？
给你链接看看成不 |
|
n***w
发帖数: 2405 | 44 额，这样阿。
前面也有个前辈说我的情况可能是出现了soluble aggregates。
嗯。GST是一定要切掉的。。。
看来真得换载体阿。。。
＝＝ |
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C*******e
发帖数: 4348 | 45 这个现象很有意思哈
为什么蛋白在柱子上变性了反而不容易被洗下来？
soluble aggregation是什么意思？ |
|
o*****r
发帖数: 156 | 46 most likely yes
kinetic |
|
o*****r
发帖数: 156 | 47 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, |
|
y******8
发帖数: 1764 | 48 No only for hematopoietic system. Basically, you can screen for any
phenotype, but use soluble molecules instead of genetic methods. By this way
, you would directly get drug candidates, not just drug target candidates.
The transparency of fish embryo is overstated. If you want deal with any
late-onset disease, the adult fish is not very transparent. |
|
W*****o
发帖数: 1780 | 49 Vector pET22b is good at expression of soluble proteins with disulfide bonds
in E.coli without any tag. |
|
v**********m
发帖数: 5516 | 50 Sadly, the truth is that the protein you are working on is likely to
precipitate out by itself. The GST tag increases the solubility of the
fusion protein.
Man, doing protein expression is nothing more than gambling or trading stocks.
割肉吧。
3) |
|
