p******6
发帖数: 410 | 1 最近要弄些临床标本提DNA做array-CGH.有些case只有用过的FISH slides available,
有高手能指教一下这些能用来提DNA做CGH吗？FISH probes所在区域我们分析时会考虑
进去的。这些片子当初都denature过，不知道这种变性后单链的DNA好不好提取？ |
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c********r
发帖数: 189 | 2 打算做一个transcription factor的 co-IP，这个蛋白主要在核内表达，表达量不高，
RIPA buffer提的话，这个蛋白不溶解，还在pellet内。可以用高盐和denature来提，
但是就影响protein interaction了。
问问大伙，用什么方法裂解细胞或细胞核比较好？
很多paper中看到，cryolysis的方法比较好，但是实验室没有相关的设备。 |
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o******p
发帖数: 55 | 3
IP.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Samples can be separated on a gel and subsequently cleaned up to remove SDS.
HCl
complexes?
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Changing the pH works, but usually denatures proteins. I used to do elutions
with ammonium hydroxide (pH>10). It is both efficient and convenient. It
can be removed simply by speed vac.
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Filter assisted sample preparation (FASP) is a popular choice. |
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z****u
发帖数: 1007 | 4 Invitrogen 的EdU incorporation kit.
染色只需要半小时就可以完成。高度敏感，特异。不需要denature. |
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l**********1
发帖数: 5204 | 5 COLD-PCR then Agarose Gel electrophoresis
Abstract
The detection of low-abundance DNA variants or mutations is of particular interest to medical diagnostics,
individualized patient treatment and cancer prognosis; however, detection sensitivity for low-abundance
variants is a pronounced limitation of most currently available molecular assays. We have recently
developed coamplification at lower denaturation temperature-PCR (COLD-PCR) to resolve this limitation.
//www.ncbi.nlm.nih.gov/pmc/articles... 阅读全帖 |
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b****r
发帖数: 17995 | 6 你们都没有好好读文章
它说了，不用denature，就是直接读双链DNA |
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b******n
发帖数: 4225 | 7 跑电泳时加点SDS在loading dye中denature DNA/protein试试看能否load进well？ |
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s******s
发帖数: 13035 | 8 //nod.
elisa/wb都是denature的，flow/stain是接近天然构想，有些antibody只能识别变性或
者天然表位 |
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x*****o
发帖数: 441 | 9 一种蛋白, 提纯之后refold然后保存在-80, 之前那个人保存的体积很大, 所以已经解冻4次了. 我现在要用这个样品.
反复多次解冻会造成什么问题,是降解还是denature, 还是两者都有? |
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i***0
发帖数: 160 | 10 If you dilute the high concentration protease to low concentration its
activity increase, then the decreased activity at high concentration is
reversible, which is good. It might due to the storage buffer you use for
concentration not friendly for the activity of the enzyme, or reversible
oligomerization that enclose the active site.
If you dilute the high concentration protease and can not restore the
activity you have seen while it is at low concentration, it is possible,
that it is aggregated... 阅读全帖 |
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b**********8
发帖数: 349 | 11 新鲜的肿瘤组织，很宝贵，用RIPA lysis buffer提取总蛋白，请大家推荐个可靠地保
存方法，
1）直接冻于-80？or
2）添加 SDS loading dye之后冻于-80？or
3）添加 SDS loading dye并denature之后冻于-80？
先行谢过 |
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m******p
发帖数: 67 | 12 western for a 25kd protein. using serum.
but the band is clearly at 50kd.
seems dimer, but in sds-page, proteins are denatured, so should not form
dimmer. any suggestions?
thank you very much. |
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K*F
发帖数: 120 | 13 请您转发 z******[email protected]
万分感谢！祝您身体健康，财源广进！
Biochim Biophys Acta. 1988 Apr 28;954(1):1-13.
The inducible trimethylamine-N-oxide reductase of Escherichia coli K12:
biochemical and immunological studies.
Silvestro A, Pommier J, Giordano G.
Source
Laboratoire de Structure et Fonction des Biomembranes, CNRS, Marseille,
France.
Abstract
The inducible trimethylamine-N-oxide reductase which migrates on non-
denaturing polyacrylamide gels with an RF of 0.22, has been purified from
the soluble fraction o... 阅读全帖 |
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S**********e
发帖数: 1789 | 14 Vanderbilt University School of Medicine
Lopez, Carlos F , Ph.D. Assistant Professor of Cancer Biology
Publications
Giovambattista, N, Lopez, CF, Rossky, PJ, Debenedetti, PG. Hydrophobicity of
protein surfaces: Separating geometry from chemistry. Proc Natl Acad Sci U
S A, 105(7), 2274-9, 2008. PMCID:2268126
Lopez, CF, Darst, RK, Rossky, PJ. Mechanistic elements of protein cold
denaturation. J Phys Chem B, 112(19), 5961-7, 2008. PMCID:2268126
Lopez, CF, Nielsen, SO, Srinivas, G, Degrado, ... 阅读全帖 |
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G*****h
发帖数: 320 | 15 如果primers 的特异性很好，没有杂带，没有intons，那么很可能就是target gene 本
身的拷贝数就很低。
如果物种的 genome 已经测序过的，可以看看这个基因的拷贝数。也可以算一下一个细
胞的 DNA 大概是多少 DNA，然后算算你放进去的DNA的量可以折算成几个拷贝。
一般1个bp 大概 10^(-9) pg。1 Mb gDNA ～1 fg。如果一套基因组大小是 1 GB，那么
～1000 fg ＝ ～1 pg。如果你放了 10 ng，单拷贝的基因就是 10^4 拷贝。
当然 genome PCR 的条件也需要 optimized。比如先要 95 C denature 5 min，前面的
一些 cycles 的annealing 和 extension 的时间长些，等等。
再就是根据你 amplify plasmid的结果（slope），看看你的 PCR amplification 的
efficiency。如果 efficiency 很低，那么大genome 里面的低拷贝的基因就更需要多
个 cycles。 |
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a******a
发帖数: 68 | 16 搜到一本书，里面如是说：
two reasons: the desired protein may be moderately absorbed to the column so
that its elution occurs after Vt in a very broad peak that is difficult to
distinguish from noise in the baseline. If this is the case, a protein
solubilization agent such as a nonionic detergent or a modest concentration
of a protein denaturant should be added to the chromatographic solvent. A
second possibility involves the dissociation of a functional protein complex
into discrete proteins of different M... 阅读全帖 |
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r*********g
发帖数: 1160 | 17 谢谢回复，那在剧烈摇动时产生热量会不会denature protein。
我看了youtube的视频，理解为摇晃完放到冰上？
我想买50ml的adapter，那么裂解50ml的culture也只用40秒就可以了么？
如果有经验，求帮忙解答，先谢过了。 |
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l****y
发帖数: 398 | 18 If it works in cell lysate, that should be good enough. Thanks |
|
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l****y
发帖数: 398 | 20 but the kinases are still active. i want them all dead. |
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R****n
发帖数: 708 | 21 My tissue sample was homogenizd by micropestle in RIPA buffer+protease
inhibitors. The serial dilution results confirmed what you said 100ug/well,
cause my 1mg/well input showed strong signal from ERK and pERK both. I
boild my sample for 5 mins to denature proteins, not sure about your protein
. I am having some trouble in Standard curve now,but I will find out
tomorrow. |
|
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d****7
发帖数: 109 | 23 try ImmunoCruz secondary ab from Santa Cruz
it only recognise native IgG, not denatured IgG, so you won't detect heavy
and light chains |
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b******n
发帖数: 4225 | 24 嗯，一般DnaK和DnaJ是一起的，用ATP/Mg++也许见效
实在不行试试denatured soluble protein或者glycerol等偏方
而GroEL用ATP/Mg++比较好解决 |
|
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m*********D
发帖数: 1727 | 26 (ZT)
Salt concentration
The phosphate groups are moderately strong acids, and thus they are always
ionized at physiological pHs. The two oxygens are equivalent, so they share
the single negative charge. These negative charges tend to blow the double
helix structure apart, which is perhaps why I haven't included them in the
forces that hold the helix together. Why is the helix stable?
Inside the cell, the monovalent salt concentration is about 0.15 M. The
major cation in the cell is K, while i... 阅读全帖 |
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k*****n
发帖数: 323 | 27 这个应该不是污染，我做illumina library的时候也遇到过这种情况，而且很常见。怀
疑是末端序列在buffer里denature然后互相anneal了，出来的都是1* 2* 3*的片段，测
序结果正常 |
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s********x
发帖数: 472 | 28 Has anyone use it for co-ip experiment? I just feel it generate too much
foam that may denature the proteins. |
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i***l
发帖数: 1656 | 29 i am telling you the trick i used
you are questioning me about my trick
what do you want to hear from me?
before asking"3-4M urea会不会太高了" have you check why use urea and what's
the usual range of urea used in denaturing buffer?
what's the conc. you think is better? |
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r********n
发帖数: 164 | 30 Did you use denature gel? RNA gel? Normal DNA gels may not give you good
estimation on the size. Purification I used RNAeasy kit from Qiagen, works
well. You could use classic pheno-chloroform extraction if you don't have
the kit. If your RNA coded product has fluorescence or very specific
phenotypes in your system maybe a direct functional verification is easier.
good luck |
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f**********s
发帖数: 1415 | 31 分离细胞核后，想用denature immunoprecipitation纯化一种可溶性核蛋白，包子求一
个lysis buffer配方和protocol. Thanks. |
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f*******d
发帖数: 92 | 32 这个样做其实对于磷酸化是最好了，这样可以迅速denature所有的蛋白，包括
phosphotase, 从而最大程度上preserve post-translational modification. 只是我
还是会在laemmli buffer里加上protease inhibitors, 煮完之后sonicate, spin down
, bradford，load supernatant. |
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i******1
发帖数: 21 | 33 这帖子还没沉？
这问题不清楚。到底要跑胶看啥？8M urea 不足以denature所有的protein，特别是好
多酶类，所以很多的modification你都看不到了, 比如phosphorylation,
ubiquitination等. 可以75C左右加热5~10分钟，有carbamylation 和其他
modification产生，但总量不到~5%，大部分protein还在。总之，具体问题具体分析。 |
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g*****s
发帖数: 1016 | 34 我做过实验 biotin-streptavidin断的不多 问题是streptavidin被denature了 |
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f*******e
发帖数: 628 | 35 streptavidin 从 bead 上脱落是不太可能的。
加热 80C 在正常的 buffer 里面，时间不是太长的话应该也没有问题。即使有问题也
是 denature 了，脱落是不应该的。 |
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m*********D
发帖数: 1727 | 36 这这这。。。怎么听起来象小伙伴打架，打不过了就找爹爹一样。。。。：）。你老板
要是新的AP,bench上的你可以听听。拿到tenure后你就随便听听。我老板和学生讨论
bench上的tech,都市线咨询我的。
我的理论依据就是gel按DNA size来分的。单链DNA跑胶的快慢会和二级结构有关，只能
用denatured gel,但双链基本是按size来分的。我这个wt和mutant是九百多个Nt里面中
间有十来个nt变化，应该基本上是维持双链。我还每看有没有G：T配呢，要有几个GT配
，那就差别更小了。 |
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i***l
发帖数: 1656 | 37 agree with above,maybe oligomer due to non specific hydrophobic interactions
, which is not uncommon among transmembrane proteins.
4-6M urea, try to completely denature your sample,
however, if oligomer forms, sample usually runs in a ladder pattern, say,
dimer, tetramer, etc. if you only see two major bands, one monomer, the
other ~4x or 6x apparent molecular weight, it's still a little weird to me. |
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K******S
发帖数: 10109 | 38 heat --> ice cold (stays denatured)
vs
heat --> gradually cool down (might 复性) |
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M******g
发帖数: 152 | 39 Try NEB Q5® Site-Directed Mutagenesis Kit https://www.neb.com/products
/e0554-q5-site-directed-mutagenesis-kit). It is much much better than the
quick change. They also have a web tool to help you design the primers: http://nebasechanger.neb.com/. Very convenient.
Make sure to use their comp cells. if you use comp cells from other
companies, dilute your final rxn (1:3), then do the transformation.
If your plasmid is big such as 8 kb, I normally double the denaturing time
as NEB sugge... 阅读全帖 |
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B***v
发帖数: 113 | 40 I'll check it out. Never used it. But sounds so good based on your
experience.
Denaturing time doesn't seem to be an issue as other mutations worked using
the same template.
春节快乐！
products |
|
发帖数: 1 | 41 1) I have no comments, but Dr. Chou has mentioned he was able to reproduce
it.
2) DNA band and migration are easily alternated by sample ionic strength and
buffer. The reason lies in the mechanism of DNA molecule electrophoresis is
so different from SDS-PAGE. DNA band shift is not a unusual result if DNA
sample denature or conformation is not consistent because DNA molecules
migrates on a electrical filed by the negative charge of its own phosphate
groups of which the charge is easily altered by... 阅读全帖 |
|
发帖数: 1 | 42
The denature process and buffer ionic strength of DNA sample will cause DNA
band migrates at an unexpected rate or at un-even shapes. Do you understand
why DNA
migrates under a certain pH and electrical filed. DNA migration, diffusion
and dispersion are three factors governing the final electrophoresis result
(DNA migration profile)
This is a whole academic issue and how it is running right now. If the rule
changes, I guess the quality of scientific research will be improved.
However, who is go... 阅读全帖 |
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n**********g
发帖数: 196 | 43 1) Do you understand statistics?
2) you need to address why they conflict with each other. (Fig 4a and 4e).
You need to read others’ question first.
The denature process and buffer ionic strength of DNA sample will cause DNA
band migrates at an unexpected rate or at un-even shapes. Do you understand
why DNA
migrates under a certain pH and electrical filed. DNA migration, diffusion
and dispersion are three factors governing the final electrophoresis result
(DNA migration profile)
3) you need ... 阅读全帖 |
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g*********e
发帖数: 150 | 44 Formaldehyde, glutaraldehyde, alcohol, etc. does not "quench" fluorescence.
Instead, it denatures the GFP such that it no longer functions and
fluoresces. Any remaining fluorescence is either non-specific (many proteins
auto-fluoresce after fixation) or it is a few GFP molecules that retain
their original conformation. If you fix the tissue, then as Ann Cantereau
states, you MUST use an anti-GFP antibody to detect the GFP. At that point,
you are "seeing" the GFP by whatever tag you have on the s... 阅读全帖 |
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p**********m
发帖数: 472 | 45 如果用reverse phase sorbent, 糖应该是不会retain的, 蛋白如果不denature的话,
也不会保留的吧. |
|
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p***r
发帖数: 533 | 47 如果没有的话，可不可以用denatured ethanol代替？ |
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t******e
发帖数: 196 | 48 J Mol Biol. 1962 Jul;5:109-18.
Determination of the base composition of deoxyribonucleic acid from its
thermal denaturation temperature.
MARMUR J, DOTY P. |
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s**********8
发帖数: 25265 | 49 那hplc不成啊 organic会denature你的protein. 你得走gravity size exclusion |
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y*****s
发帖数: 1047 | 50 not very likely, antibiotics and hormones are mostly small molecules.
They don't get denatured by heat. But some of them may degrade under heat. |
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