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全部话题 - 话题: elute
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t****g
发帖数: 120
1
来自主题: Biology版 - IP的elution
请问大家做IP最后elution一步都用什么条件? 我们实验室的protocol是用将最后一步
wash的buffer remove掉几乎没有,然后加15-20ul 5x SDS loading buffer在沸水里放
10分钟。 这时间是不是太长了? 温度是不是太高了?
好奇问一句,有不用高温加热的吗? 听做proteomics的说,加热会产生抗体的片断,
干扰做mass spec。 如果想elute出protein(s)做MS,该用什么elution 条件呢?
n***w
发帖数: 2405
2
来自主题: Biology版 - 关于GST elution buffer
一般的Elution buffer都是50mM Tris-Cl和10mM的reduced glutathione, 然后pH 8.0。
如果用50mM或者100mM的reduced glutathione呢?需要相应增加Tris-Cl的浓度和pH吗?
谢谢。
c********r
发帖数: 189
3
来自主题: Biology版 - IP的elution
1) crosslink your antibody with the beads
2) elute with 0.1M glycine(pH 2.5)
g*********5
发帖数: 2533
4
来自主题: Biology版 - IP的elution
for me, boiling 3min is enough for the western.
if you want to MS, elute should be better.
j*****a
发帖数: 92
5
阴离子交换色谱 Q Sepharose Fast Flow (GE Healthcare Life Sciences)pH
stability Working Range 2-12 .用1 M NaOH 洗,用纯水洗到pH 7, 换TrisHCl
buffer pH 5.5 平衡, elute pH 变成9.洗了一天还是pH 9. 有人遇到过吗?
j*****a
发帖数: 92
6
阴离子交换色谱 Q Sepharose Fast Flow (GE Healthcare Life Sciences)pH
stability Working Range 2-12 .用1 M NaOH 洗,用纯水洗到pH 7, 换TrisHCl
buffer pH 5.5 平衡, elute pH 变成9.洗了一天还是pH 9. 有人遇到过吗?
A*****8
发帖数: 8590
7
Report Information:
Publication date: 11/07/2012
Number of Pages: 145
Report Details:
In 2011, the medical device market worldwide was worth $309bn, with high
single-digit growth from the year before . Despite being a vast and mature
market, there are number of sectors likely to experience fast growth during
the next 10 years. Much of this growth is linked to the prevalence of a
number of diseases worldwide, as well as an aging population. The rise in
neurological disorders will drive both the d... 阅读全帖
b*********b
发帖数: 64
8
来自主题: Biology版 - 请教:BAC Maxi Prep的问题
I use QIAGEN Plasmid Midi kit. 100ml culture could give about 30-50ug. I
tried Qiagen's large construct kit.But in my hand, the midi kit is much much
better than the large construct kit, and also takes much less time.The
following is the protocol I got from Qiagen.
User-Developed Protocol:
Isolation of BAC DNA using the QIAGEN® Plasmid Midi Kit
This procedure has been adapted by customers from the QIAGEN® Plasmid
Midi Kit Protocol.It has not been thoroughly tested and optimized by QIAG... 阅读全帖
v***a
发帖数: 1242
9
来自主题: Biology版 - 关于co-ip的!
我也有类似的co-ip不work的困扰。
请问你说的:
“Crosslink 1st antibody to beads and elute co-purified protein complex from
beads after washing (instead of boiling beads) to reduce background”
怎么个crosslink法?我是加了1st Ab overnight再加beads的。
还有elute我是用的SDS sample buffer,然后就boil,100 C,10min。boiling beads
和elute是两个独立的过程呀,为啥elute可以取代boiling?是我理解错你的意思了吗?
关于ECL plus,我觉得用ECL曝光久一点,貌似也跟plus一样的效果,而且plus还会令
blot颜色深浅不均一,不知道你会有这个问题吗?如果有,怎么解决比较好?
谢谢回答。

of
l*******1
发帖数: 128
10
I think you might need to do consecutive affinity column/pull down. Let's
say you tag your A, B, C, D with GST, V5, His, and HA.
You can start with GST-fused A to pull down the A-containing complexes.
Then pass the elution to V5-tag beads, elute the presumed A&B containing
complexes with V5 peptide. Pass the A&B containing complexes through His-
tag beads, elute the ABC complex with imidazole.
Use Western to probe the original sample and each elution for those 4 tags.
Of course, you can chang... 阅读全帖
f*****s
发帖数: 104
11
最近用GE healthcare的glutathione sepharose 4B resin purify a couple of GST-
tagged transcription factor DNA binding domains. The protein were bound to
the beads with high efficiency. However, when I used glutathione buffer to
elute the GST-protein off the beads, they can't be eluted. I tried 10 mM ,50
mM and 100 mM glutathione. To make the buffer, I diluted 1 M Tris (pH 7.5
at room temperature) to 50 mM, and added glutathione powder. There was also
2 mM DTT in the buffer. The elution was carried out... 阅读全帖
M****m
发帖数: 2142
12
来自主题: Biology版 - 还是一些Co-IP问题
我的目的是identify interantion partners and send for sequencing,这样对蛋白
量的要求就比较大不像western似的一点就够了。大家一般elution的时候都是多少ul?
还有我用的FLAG的beads,protocol建议3种,我选了low acidic的buffer,这样不管什
么在beads上都一起给洗脱下来了,不过我有control还好可以区分哪些unspecific的
binding,我用了100 ul elution buffer,那这样的话量太大没办法跑胶,浓缩可以有
几种方法,一是用centricon binding protein然后加少量的buffer elute,二是
lyophilization,基本上就是减少水的含量,这样盐浓度大幅度提高,不知道好不好,
还有就是我先都加dye煮了,然后再vaccum dry减少水分以降低volume便于跑胶,哪种
方法更可行一些?
e*****n
发帖数: 15
13
哎,都博士二年级了,还问这样的问题,真不好意思
我是用qiagen blood & tissue kit 提brain tissue的DNA,~ 20 mg, handbook上面
说 brain tissue的话25mg 的DNA 产量 15 ~ 30 ug, 那理论上我应该得到12 ~ 24 ug。
但是我做了几次,最好的那次是200ul elution buffer,浓度 ~ 30 ug/ml, DNA 总量
是6 ug。
今天又做了2个sample,200 ul elution buffer,浓度只有10 ug/ml, 再用新的100ul
elution buffer, 浓度~10 ug/ml, DNA 总量大概只有3 ug。跑了一块胶,可以确定是
有DNA的。
实在想不出来是什么原因。 我disrupt tissue的时候是用23 G的needle做的,然后加
proteinase K消化了6个小时,液体很澄清,应该不会是消化的不好吧。很简单的实验
,就是做不好,哎。。。。
Sample比较珍贵,不敢再做了。。。希望大家能帮我一下啊,谢谢大家!!
s********8
发帖数: 619
14
来自主题: Biology版 - 求推荐胶回收试剂盒
好像是啊,还真不知道qiagen就有这个东西.不过这个beads回收的elution体积一般都挺
大的是吧?还是想找一个好用的kit.Zymo 的kit可以elute 在6ul elution buffer里,但
是就是有时候work的不是很好.
p******i
发帖数: 1092
15
来自主题: Biology版 - 免疫沉淀失败,求助
你用DSS是因为不想在elution里看到IgG吗?
如果你那个蛋白的size离heavy chain light chain不近,
你先试试不用cross-link做IP好了……
你elution volume是多少?都跑胶了吗?
如果volume大可以concentrate一下,全跑
这个KIT你可以自己order各个试剂,elution buffer也可以找到recipe啊
另外,顶楼上,膜蛋白不好做,搜一搜别人的paper,看用的是什么条件
IP的抗体也要多试几个……
300-400ug有点少啊,多弄点sample啊
q***7
发帖数: 144
16
来自主题: Biology版 - Help: mouse genotyping by sequencing
Qiagen做为柱提取DNA的老大,从来没有想着怎么改进他们的产品。他们柱子elute出来
的DNA大都含有洗液,尤其是Gel Extraction, PCR purification, and RNAeasy kits
。你用个连有真空的小枪头在最后一步要elute你的DNA时在柱子里面的那个垫圈上吸吸
看有什么东西。你会看到小枪头里都是洗液。Green BioResearch公司,就想到了这一
点,垫圈上设计了个小洞,这样就不会有洗液留在上面了。这样你elute出来的样品里
就不会含有洗液,而不会抑制后面的酶的活性了。
你可以看一下这个连接
http://www.greenbioresearch.com/gel-extraction-pcr-purification

QIAGEN
g*********5
发帖数: 2533
17
来自主题: Biology版 - 核蛋白的Co-IP的 Protocol
Isolation of nuclei for the co-IP and whole ChIP protocols is based on the
methods of Gendrel et al. (2002, 2005), Johnson et al. (2002), and Nelson et
al. (2006).
METHOD
For extraction and co-IP of nuclear proteins, see Steps 1-39 (Fig. 1). For
the ChIP procedure, see Steps 40-69 (Fig. 2).
Figure 1.
View larger version:
In this page
In a new window
Figure 1.
Flowchart for the timeline and organization for co-IP of nuclear proteins
from Arabidopsis seedlings.
Figure 2.
View larger versio... 阅读全帖
c******m
发帖数: 884
18
来自主题: Chemistry版 - pKa 问题,请教各位大牛
Thank you for the reply. Do you mean triethylamine for TEA? If so, I didnot
use any TEA in the previous wash.
The ion exchange (IX) solid phase (SP) contains a linker-piperazine with pKa
of 6, so I load the orotic acid (ORA, pKa1=2.8) with a pH 4.4 (half of 2.8+
6) solution to make sure both SP and ORA are ionized. Then I use 2%Formic
Acid/water (pH=2) to wash (should not elute any ORA out), and then use NH4OH
in MeOH/ACN to elute ORA. But the result shows the acidic wash step elute a
lot ORA. S... 阅读全帖
C*I
发帖数: 4736
19
一直在误导,现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人,必须
经过一个中间宿体性的其它动物才能传染给人。所以,病毒发生后,就故意误导全国人
民去海鲜市场找证据,找其它野生动物的麻烦。 而且还是病毒所去找的,找完了还装
模做样化验呀,分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
,一直不敢说,说了其它相关已经在人就会去早那种动物监测。 所以压根不说,打马
虎眼。
事实上,早在2013 年,就是这个武汉病毒研究所,已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒,其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体,并能够感
染人的细胞。对此新发现,武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”,以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说,从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus, 可以不经过
其它受体/宿体,而直接传染给人。 他们... 阅读全帖
C*I
发帖数: 4736
20
一直在误导,现在还在误导。 说说明corona virus 不可以从蝙蝠直接传染给人,必须
经过一个中间宿体性的其它动物才能传染给人。所以,病毒发生后,就故意误导全国人
民去海鲜市场找证据,找其它野生动物的麻烦。 而且还是病毒所去找的,找完了还装
模做样化验呀,分离呀什么的。最后把责任全部推给了海鲜市场的动物。可是那种动物
,一直不敢说,说了其它相关已经在人就会去早那种动物监测。 所以压根不说,打马
虎眼。
事实上,早在2013 年,就是这个武汉病毒研究所,已经从来自云南的蝙蝠身上所携带
的corona virus中分离出第一株蝙蝠SARS类似样的冠状病毒的活病毒,其中就包含了类
似于S类型的基因。从而证实这株病毒能够使其接受和SARS病毒相同的受体,并能够感
染人的细胞。对此新发现,武汉病毒所还把它以武汉病毒研究所的英文简称命名“WIV1
”,以彰显这一发现的重要价值和属于自己第一个发现的巨大研究成果。这个成果刊载
于2013年11月的《自然》杂志。
就是说,从云南弄回来的这种蝙蝠所携带的类似于sars的 corona virus, 可以不经过
其它受体/宿体,而直接传染给人。 他们... 阅读全帖
C*I
发帖数: 4736
21
Published: 30 October 2013
Isolation and characterization of a bat SARS-like coronavirus that uses the
ACE2 receptor
Xing-Yi Ge, Jia-Lu Li, Xing-Lou Yang, Aleksei A. Chmura, Guangjian Zhu,
Jonathan H. Epstein, Jonna K. Mazet, Ben Hu, Wei Zhang, Cheng Peng, Yu-Ji
Zhang, Chu-Ming Luo, Bing Tan, Ning Wang, Yan Zhu, Gary Crameri, Shu-Yi
Zhang, Lin-Fa Wang, Peter Daszak & Zheng-Li Shi
Nature volume 503, pages535–538(2013)Cite this article
Abstract
The 2002–3 pandemic caused by severe acute respirator... 阅读全帖
l***d
发帖数: 1798
22
FOR IMMEDIATE RELEASE
National Academy of Engineering Elects 66 Members and 10 Foreign Associates
WASHINGTON — The National Academy of Engineering (NAE) has elected 66 new m
embers and 10 foreign associates, announced NAE President Charles M. Vest to
day. This brings the total U.S. membership to 2,254 and the number of forei
gn associates to 206.
Election to the National Academy of Engineering is among the highest profess
ional distinctions accorded to an engineer. Academy membership honors th... 阅读全帖
s*********n
发帖数: 149
23
来自主题: Medicine版 - 请教心脏支架的选择
劝你用美国货,Abbott 的Xience, or Medtronic的Endeavor. drug eluting stent确实
缺少长期试验数据,可是也已经在市场上近10年.前一阵的那篇关于bare metal比drug
eluting好的文章,当时引起了巨大的反响,可是几年下来,事实并非如此.
国产的好像叫火鸟.不过国产的质量不好说.毕竟和FDA相比,SFDA需要的批支架的试验数
据不可同日而语.现在严格多了,可是已经上市的就上市了.antiplatelets 要和阿斯匹
林一起吃.防止血栓.
d*****r
发帖数: 2583
24
☆─────────────────────────────────────☆
haoyun (福娃) 于 (Thu Dec 18 17:35:33 2008) 提到:
要抽100kb左右的BAC DNA用于end sequencing,哪个公司的kit比较好?
谢谢指教!
实验室里普通的plasmid我们都用Qiagen的midi prep kit
也可以用于BAC DNA extraction吗?
我以前用土法抽过BAC clone,恶梦啊
100ml culture然后用Qiagen midi kit够不够sequencing?
我以前土法抽BAC累不说,sequencing结果非常不好
☆─────────────────────────────────────☆
longwoodwalk (想做点啥) 于 (Thu Dec 18 17:41:14 2008) 提到:
用qiagen的high-speed maxi prep kit. 效果非常好。唯一注意的是,在elute你的DNA
之前,将Elution buffer (QF?) preheat到65-7
l*******1
发帖数: 128
25
来自主题: Biology版 - 请教高手如何胶回收17Kb质粒
I generally have no problems in midi preps for our transgenic constructs
which are usually around 15-21kb.
However, as suggested in the Qiagen protocols, you can pre-warm the elution
buffers to 65C to increase the yield of large plasmids. You could also try
to elute the DNAs slowly to increase the time needed for large DNAs to be
dissolved off the columns.
s****a
发帖数: 436
26
Purification of biotinylated proteins on streptavidin resin: A protocol for
quantitative elution。
For elution of biotinylated proteins, 500 mL
release solution (2% SDS, 30 mM biotin, 50 mM phosphate,
100 mM NaCl, 6 M urea, 2 M thiourea, pH ,12)
were added to the resin and incubated for 15 min at RT,
followed by incubation for 15 min at 96C. Afterwards, the
resin was pelleted by centrifugation for 5 min at 16 100g
a*****n
发帖数: 2835
27
来自主题: Biology版 - 有人用sulfo-NHS-LC-biotin吗?
1, 标记完之后要使劲洗,洗掉free的biotin
2,binding完了之后怎么elute的?elute效率可能有问题
w******e
发帖数: 1187
28
来自主题: Biology版 - 有人用sulfo-NHS-LC-biotin吗?
very good point! I used zeba column to get rid of free biotin, but
never really tested the efficiency (in fact now I think of it,
it'll be hard to measure how much free biotin is left... do you
just do multiple column purification to make sure of that?)
2. sorry can you elaborate on the elution part? I don't quite
understand the purpose for eluting after binding w/ beads.
C***Y
发帖数: 61
29
来自主题: Biology版 - 关于co-ip的!

PBS or washing buffer can be used for incubating Ab with beads;
I have never done preclear;
Glycine solution (pH~2.8) can be used for elution. After elution, add equal
volume of 1 M Tris, PH7.5 (for
neutralization) and equal volume of 3 X SDS loading buffer.
n****n
发帖数: 165
30
Just using WB or CB detects your protein and this protein should be concentrated in the late eluting volume after protein complex eluted first. You can follow company protocols that should include the detailed information and steps.
I*****y
发帖数: 6402
31
I think it really depends on your buffer used for your IEX elution. I don't think a
sizing column is sensitive to salt.
If you still have concern with the salt from your IEX elution, just try
changing the buffer to low salt when you concentrate your proteins.

column
e****s
发帖数: 1125
32
来自主题: Biology版 - 还是一些Co-IP问题
你的蛋白样品用90% methanol precipitate,然后用合适体积的Sample buffer复溶就
好了。
我感觉能用Sepcific elute比其他方法都好,应该用Flag peptide Elute最好。
C******8
发帖数: 602
33
来自主题: Biology版 - 问个magnetic beads的问题
听版上人说做CoImmunoprecipitaion用magnetic beads可以减少nonspecific
手里刚好有active motif ChIP kit里的magnetic protein G beads
我可不可以用这个beads conjugate antibody,然后去拽cell lysate的antigen呀。心
里想差别也就是ChIP里protein先crosslink了DNA嘛。。。
washbuffer还有elution buffer有什么要注意的么?Elution难道直接加sds sample
loading buffer煮一煮,dissociate下来protein到buffer里然后load?
如果新买其它beads还得等几天,试试可不可以呀?有没有前人经验。。。
n******5
发帖数: 21
34
来自主题: Biology版 - 问个magnetic beads的问题
我觉得M-beads, 主要是方便,和 minimized carry-over therefore increase
recovery. if you want to minize non-specific interactions, try follow some
naked beads to absorb them.
但是缺点就是 binding capacity 很低。
Wash buffer:建议加一些non-ionic detergent, like Triton100
Elution buffer: SDS+boil will get everything off, but all in denatured form,
it is perfectly fine if you directly go on to SDSPAGE.
if you want to maintain functional activity, I would suggest using low pH
elution (Try pH2.0 glycine, use Tris-pH8 to neutra... 阅读全帖
a****d
发帖数: 1919
35
My experience:
heating up the elution buffer;
after mixing the eluted product with isopropanol,store at -20 for couple
hours,or overnight,then centrifuge.
s******y
发帖数: 220
36
来自主题: Biology版 - 请教IP怎么控制input的量
做immuno-precipitation, elution的步骤是直接把蛋白-抗体结合的beads放到gel
loading
buffer里面煮, 然后上样。
不能测量蛋白浓度的话,怎么掌控load的量呢?
我做了两个control,尽可能treat他们一样,可是差别貌似很大,请问能elute以后测
浓度再load
吗?
多谢啦!
C*******e
发帖数: 4348
37
来自主题: Biology版 - Glutathione Agarose求教
前辈分享个方子?
lysate/binding buffer
和elute buffer
你都用同一个么?
我看说明说binding最好pH 7.4,elute pH 8.0
可是我试过
效果并不比两个都是pH 8.0好多少
V********n
发帖数: 305
38
来自主题: Biology版 - Ab purification from hybridoma cells
俺用的方法。也是用Protein G的办法,Buffer 用Pierce的Binding Buffer和Elution
Buffer。得到的Elution,直接用Millipore的Spin Column清洗加浓缩,选大一些的Cut
(比如50KD),避免小片段污染。
看你的胶,有小片段(~20 KD),可能是蛋白降解,也可能是小片段污染。另,高
分子处还有条带(~200KD),可检查蛋白变性是否彻底。
E**********1
发帖数: 73
39
来自主题: Biology版 - Co-IP 求助
做蛋白蛋白interaction.蛋白A MW 255KD, 蛋白B MW 58KD,蛋白C MW 229KD.每个蛋白
有GFP, Flag等tag.Overexpression 做Co-IP.
先做A和B的相互作用,用protien A beads,IP后boil.先IP B, WB A.对照有(1)non-
transfected, (2)only transfected with A, 试验组(3)transfected with A and
B. IP后发现(1)没有A,(2)中有weak band of A,(3)中有strong band of A.
然后怀疑A蛋白太大,可能和beads或者antibody for IP有non-specific结合,所以(2
)中有weak band of A.之后change strategy: IP A, WB B.后来发现有同样情况。
请问下大家有没有发现蛋白和protein A beads non-specific结合的情况?
然后又换flag-crosslinked beads 做,IP B, WB A. 用Flag-pept... 阅读全帖
v***a
发帖数: 1242
40
第一次做这个,还请大家多多指教。
因为目标蛋白大多在inclusion body里,所以单独分离出并用6 M guanidine溶解后过
柱。洗液1、2、3都含有8 M urea。洗液1不含imidazole,洗液2含10 mM imidazole,
洗液3含25 mM imidazole。最后用含200 mM imidazole的buffer来elute。
纯化后跑胶,发现elute后的蛋白不干净,跟小试相比有很多杂band,不过目标蛋白的
band还是清晰的。问题是洗出液3中含有非常清晰且干净的一条band,赫然在目标蛋白
处。
这是为什么呢?是代表我的目标蛋白在25 mM imidazole时就会被洗脱吗?
谢谢!
m****M
发帖数: 360
41
IP(Immunoprecipiation)elute下来的蛋白,用MS来鉴定新相互作用蛋白。
1。Elute下来的大部分是IgG,其他蛋白complex只是占1-2%,这1-2%的蛋白
是否都能被检测到, 不管含量高低?
2。就这1-2%中的一个蛋白来说,是不是这个蛋白的所有的序列都能被测到(几个
不同酶消化的肽段拼接在一起)?
谢谢指点。
c********b
发帖数: 363
42
不知道大家是怎么纯化抗体的,挂IgG的proein A柱子得到的肯定不纯。
如果是用antigen-affinity purification就会好很多。只是有时候那个his-tag还是
会干扰。一个师兄教我先用一个带HIS6的无关蛋白把non-specific的先去掉(比如
sunnyday最喜欢的actin就被我加了his6来干这事),然后再来做antigen-affinity。
如果是intein-tag提出来的这一步都可以省掉,只是intein得到的蛋白量好像没有his
-tag提出来的多。
要提到的是这个protocol在我手上得到的抗体浓度很低,我一般用1:100稀释,但是很
特异。还有就是不太适用于IP。
这个方法不用挂柱子,跑SDS-PAGE,直接转到膜上,block之后先把血清和actin-
his6(in my case)的膜放2hr,然后上清拿出来和block过的antigen的膜泡2hr,
Wash with 1XTBS(contains 0.02% sodium azide) twice, 10 min each,Elute
with glycine(pH3)... 阅读全帖
c********b
发帖数: 363
43
不知道大家是怎么纯化抗体的,挂IgG的proein A柱子得到的肯定不纯。
如果是用antigen-affinity purification就会好很多。只是有时候那个his-tag还是
会干扰。一个师兄教我先用一个带HIS6的无关蛋白把non-specific的先去掉(比如
sunnyday最喜欢的actin就被我加了his6来干这事),然后再来做antigen-affinity。
如果是intein-tag提出来的这一步都可以省掉,只是intein得到的蛋白量好像没有his
-tag提出来的多。
要提到的是这个protocol在我手上得到的抗体浓度很低,我一般用1:100稀释,但是很
特异。还有就是不太适用于IP。
这个方法不用挂柱子,跑SDS-PAGE,直接转到膜上,block之后先把血清和actin-
his6(in my case)的膜放2hr,然后上清拿出来和block过的antigen的膜泡2hr,
Wash with 1XTBS(contains 0.02% sodium azide) twice, 10 min each,Elute
with glycine(pH3)... 阅读全帖
S******e
发帖数: 393
44
谢谢!
我做了两次,基本是一致的结果,目前还在做。
用的是sigma的M2 flag agarose beads,
600-1000ug总蛋白做IP,pre-clearance with IgG beads for 3hrs,
add flag beads, 4度rotate for 5hrs,
wash 3 times with lysis buffer,
then wash once with high salt(500mM NaCl) lysis buffer,
final wash with TBS,
SDS sample buffer elute(下次准备用flag peptide elute)
还请多指教。

domain
affinity
L*******e
发帖数: 2153
45
来自主题: Biology版 - 求mass spec高人指点
I am planning to prepare coIP samples and use mass spec to get a protein
binding profile. By comparing control and mutant groups, I am hoping to
find targets that are more disease relevant. I use magnetic beads for Co-IP.
Now here comes the questions:
Would it be good enough to just use SDS buffer for elution? I know this is
the case if my next step is western blotting.
If not, would an acidic glycine buffer followed by a basic buffer (Tris-HCl
) be sufficient since this only results elution o... 阅读全帖
S*********s
发帖数: 304
46
来自主题: Biology版 - 求mass spec高人指点
1.FLAG-HA double tag->stable cell line
2.Flag elute/HA elute
3.send out for MS sequencing

IP.
HCl
complexes?
y******8
发帖数: 1764
47
来自主题: Biology版 - 3X flag tag
3xFlag is about 10x stronger than 1xflag. 3xflag peptide elution is
about 50% of recovery.
If just for Mass Spec, acidic elution is good and complete.
g***y
发帖数: 205
48
来自主题: Biology版 - 请问纯化蛋白complex
我就是这么干的,把elution全部打质谱
找在wt存在的,在deletion mutant缺失的蛋白的
不过后面又用其它方法验证了

)
elution
b******n
发帖数: 4225
49
来自主题: Biology版 - his-tagged蛋白纯化问题
有没有取一些柱介质跟2X loading buffer混合之后跑电泳
如果在柱介质中看到大量目的蛋白,说明你的蛋白可能堵在柱子里面了
elution buffer需要添加高浓度蛋白变性剂才能elute下来
40kd的小带可能是molecular chaperone DnaJ
m****n
发帖数: 1066
50
来自主题: Biology版 - Direct sequencing of PCR product.
1.PCR product is about 200bp. PCR product was run on an agarose gel, cut off
from the gel and purified using a Qia quick gel extraction kit from Qiagen
. The elution buffer doesn’t have EDTA.
2.PCR primers were used as sequencing primers.
3.During the first try, some part of the sequence was readable. The
remaining was mixed with NNNN.
4.During the second try, I redid the PCR and gel-purification. All of the
sequences were NNN. The same sequencing contractor was used.
I like to make some change... 阅读全帖
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