a********g
发帖数: 558 | 1 最近做GST-tag的蛋白纯化，蛋白纯化出来后，在sepharose-４bbeads上切，elusion下
来蛋白总有杂带，一条大概７０kd，一条２７kd后者经检验发现是GST。前者未知，请
大牛们帮忙判断下是什么原因？ | b******n
发帖数: 4225 | 2 70kD band might be DnaK
it's very normal to copurify the molecular chaperone when you try to purify
those overexpressed fusion proteins
to remove DnaK, you can add 5 mM ATP and 5 mM Mg2+ into lysate and incubate
for 20 min at room temperature prior to purification (i.e., before loading
onto the affinity column)
【在 a********g 的大作中提到】
: 最近做GST-tag的蛋白纯化，蛋白纯化出来后，在sepharose-４bbeads上切，elusion下
: 来蛋白总有杂带，一条大概７０kd，一条２７kd后者经检验发现是GST。前者未知，请
: 大牛们帮忙判断下是什么原因？
| a********g
发帖数: 558 | 3 Thank you so much.
purify
incubate
【在 b******n 的大作中提到】
: 70kD band might be DnaK
: it's very normal to copurify the molecular chaperone when you try to purify
: those overexpressed fusion proteins
: to remove DnaK, you can add 5 mM ATP and 5 mM Mg2+ into lysate and incubate
: for 20 min at room temperature prior to purification (i.e., before loading
: onto the affinity column)
|
|
