z***g
发帖数: 28 | 1 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!! | b******n
发帖数: 4225 | 2 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***g
发帖数: 28 | 3 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】
: 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
| b******n
发帖数: 4225 | 4 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******g
发帖数: 3858 | 5 你的elution buffer里应该有detergent吧 | s*******e
发帖数: 1010 | 6 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***g
发帖数: 28 | 7 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***g
发帖数: 28 | 8 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***g
发帖数: 28 | 9 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。
| q*******8
发帖数: 768 | 10 why not try western blot. 烤蓝出来的条带不一定就是你要的，那些很难看清的小条
带反倒可能是你要的 | q*******8
发帖数: 768 | 11 还有你NI出来的蛋白溶液含有大量的NaCl还有大量imidazole，这些你要是不透析啊或
者过microcon之类直接上凝胶。。。结果都是不太好说的。。。 | s*******e
发帖数: 1010 | 12 Oligomerization is not bad, unless it aggregates in void volume. In fact a
lot membrane proteins exist as natural oligomer.
if
【在 z***g 的大作中提到】
: 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!
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