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about membrane protein purification

about membrane protein purification# Biology - 生物学

d*f2012-11-19 08:11

1 楼

【以下文字转载自 ChinaNews 讨论区】
发信人: fairbanks (PAI), 信区: ChinaNews
标题: 美国道路安全比中国差得多
发信站: BBS 未名空间站 (Wed Sep 15 12:14:04 2010, 美东)
如果一个地方大家开车特别客气让来让去，说明这个地方交通流量一定挺低的。要不然
，哪有那么多公共道路资源让你去送人情？连着来几个直行让左转的，路还不就瘫了？
车多路自然难行，所以就算老是吹捧美国交通秩序如何好的人们，也经常用各种恐怖夸
张的语言评论美国的大镇店比如纽约LA什么的。
数字说话，2007年，美国道路交通事故死亡人数41049人，美国人口3亿，交通死亡率为
万分之1.3. 同一年，中国美国道路交通事故死亡人数81649人，中国人口13亿，交通死
亡率为万分之0.62。中国人口占世界的22%，却只占15%的交通事故死亡人数，很了不起。
http://www.census.gov/compendia/statab/cats/transportation/motor_vehicle_accidents_and_fatalities.h

z*g2012-11-19 08:11

2 楼

Hey guys
I am doing purification of membrane proteins to get enough concentration for
NMR study, the protein was labeled by his-tag
Firstly, the protein was expressed by E. coli, lysed by several enzymes,
agents and detergent(empigen) to solublize everything, then it was purified
by Ni-NTA column, the protein shows at the right MW(about 18KD) at commassie
blue stained gel.
However, the problem is, after the second purification---ion exchange on
Capto Q(pH 8.0), the protein which we collected doesn't show at 18KD,
instead, there is a very thick band on about 23KD, we think it might be due
to the salt effect when ion exchange, or autocleavage of the protein when
dimerize? but we didn't prove our thoughts by western blot
Did this happen to anyone of you before? Please tell me if you know
something
Thank you very very much!!

a*e2012-11-19 08:11

3 楼

要是不算撞了后再碾几碾的，中国的死亡率还会低。不过那也低不过俺们公司的停车场
，车辆密度至少80%以上，死亡率为0。

b*n2012-11-19 08:11

4 楼

please upload the SDS-PAGE photos before and after ion-exchange
we may have better idea

for
purified
commassie
due

【在 z***g 的大作中提到】

: Hey guys
: I am doing purification of membrane proteins to get enough concentration for
: NMR study, the protein was labeled by his-tag
: Firstly, the protein was expressed by E. coli, lysed by several enzymes,
: agents and detergent(empigen) to solublize everything, then it was purified
: by Ni-NTA column, the protein shows at the right MW(about 18KD) at commassie
: blue stained gel.
: However, the problem is, after the second purification---ion exchange on
: Capto Q(pH 8.0), the protein which we collected doesn't show at 18KD,
: instead, there is a very thick band on about 23KD, we think it might be due

z*g2012-11-19 08:11

5 楼

效果可能不太好，明天好好照个像，marker左第一条是ni-nta纯化过的，第二条是ion
exchange纯化过的

for
purified
commassie
due

【在 z***g 的大作中提到】

b*n2012-11-19 08:11

6 楼

好像在23KD那过了Ni-NTA柱也有一个条带，比较淡而已
ion exchange之后是不是只是enrich了这个non-specific band
如果可能搜集过ion-exchange的flow through跑一下SDS-PAGE
另外一种可能是蛋白沉淀之后trap在柱子里面了

ion

【在 z***g 的大作中提到】

: 效果可能不太好，明天好好照个像，marker左第一条是ni-nta纯化过的，第二条是ion
: exchange纯化过的
:
: for
: purified
: commassie
: due

q*g2012-11-19 08:11

7 楼

你的elution buffer里应该有detergent吧

s*e2012-11-19 08:11

8 楼

ion exchange不是纯化膜蛋白的好方法，大部分膜蛋白表面都盖着detergent，很难携
带电荷去结合柱基。能不能过分子筛试试呢。

z*g2012-11-19 08:11

9 楼

Thank you pal, actually the flowthrough is the third left to the marker,
sorry for not mentioning...and the 23KD and 18KD are also in the flowthrough
( but so weak compared to the peak I collected), it's possible that the
protein was trapped, but the amount of proteins seems the same to me,
although we didn't quantify... haha, thank you very much for answering

【在 b******n 的大作中提到】

: 好像在23KD那过了Ni-NTA柱也有一个条带，比较淡而已
: ion exchange之后是不是只是enrich了这个non-specific band
: 如果可能搜集过ion-exchange的flow through跑一下SDS-PAGE
: 另外一种可能是蛋白沉淀之后trap在柱子里面了
:
: ion

z*g2012-11-19 08:11

10 楼

Thank you~ yes, u r right, there is 0.7% empigen

【在 q******g 的大作中提到】

: 你的elution buffer里应该有detergent吧

z*g2012-11-19 08:11

11 楼

yes, very good idea, recently we also plan to do size exclusion, but what if
my protein oligomerizes during the process, thank you very much!

【在 s*******e 的大作中提到】

: ion exchange不是纯化膜蛋白的好方法，大部分膜蛋白表面都盖着detergent，很难携
: 带电荷去结合柱基。能不能过分子筛试试呢。