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how to purify the degraded protein bands on the gel?

how to purify the degraded protein bands on the gel?# Biology - 生物学

w*n2013-07-20 07:07

1 楼

暑假回国参会，顺便参观了北京两所985，都是有熟人在那儿教书
发现两个人所在的系，教师都没有独立办公室
一个系是一个大房间，每位老师一个桌子
另一个系是相对小的房间，每个房间2-4位老师
只有系主任有自己独立的房间
这是普遍现象吗？

c*42013-07-20 07:07

2 楼

My protein is around 20kDa, and I see many degraded protein bands (from
10kDa to 3kDa) on the gel. I tried to wash my protein several times using
the amicon centrifugal filter at 10kDa cutoff, but I still see those
degraded protein bands on the gels. I also add proteinase inhibitor and also
do the purification on ice to prevent the degradation, but it does not help
. Does anyone know what is the best way to purify or get ride of these
degraded protein bands on the gel? thanks.

j*l2013-07-20 07:07

3 楼

不能说普遍但也不能算奇怪吧，10-15年以前可能更普遍些

【在 w*****n 的大作中提到】

: 暑假回国参会，顺便参观了北京两所985，都是有熟人在那儿教书
: 发现两个人所在的系，教师都没有独立办公室
: 一个系是一个大房间，每位老师一个桌子
: 另一个系是相对小的房间，每个房间2-4位老师
: 只有系主任有自己独立的房间
: 这是普遍现象吗？

e*s2013-07-20 07:07

4 楼

Your title is really confusing.
This could be very challenging. Because the truncated forms, especially
those with similar size to the full-length, behave very similar to full-
length protein. If most of your truncated is smaller than half of the full-
length, then it is doable. Gel filtration may helps to remove those big
truncated ones. You can also try those traditional columns, such as ion-
exchanged or hydrophobic columns.
Amacon's concentrator was not designed to do this.
I will suggest to prevent it from happening by adding protease inhibitors;
however,sometimes it is just the way it is.
If you really need full-length proteins, you can also add different tags at
each side.

also
help

【在 c*******4 的大作中提到】

: My protein is around 20kDa, and I see many degraded protein bands (from
: 10kDa to 3kDa) on the gel. I tried to wash my protein several times using
: the amicon centrifugal filter at 10kDa cutoff, but I still see those
: degraded protein bands on the gels. I also add proteinase inhibitor and also
: do the purification on ice to prevent the degradation, but it does not help
: . Does anyone know what is the best way to purify or get ride of these
: degraded protein bands on the gel? thanks.