c*******4
发帖数: 41 | 1 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. | e****s
发帖数: 1125 | 2 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.
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