小中大
GST纯化出现杂带不算是很奇怪的事,从你的电泳胶上可以看到,目的蛋白很多都流穿了,说明你的蛋白折叠不好,相当于凝胶使用过量,有非特异性结合。
另外,我看了一下说明书,下面这段话也许对你有所启示:
Multiple bands are
observed after
electrophoresis/
Western blotting
analysis of eluted
target protein.
Mr 70 000 protein copurifies
with the GSTtagged
protein
• The Mr 70 000 protein is probably a protein
product of the E. coli gene dnaK. This protein
is involved in protein folding in E. coli. It has
been reported that this association can be
disrupted by incubating the tagged protein
in 50 mM Tris-HCl, 2 mM ATP, 10 mM MgSO4,
pH 7.4 for 10 min. at +37°C prior to loading
on filter plate.
• Alternatively, remove the DnaK protein by
passing the tagged protein solution through
ATP-agarose or by ion exchange.
我曾经遇到结合不好的GST融合蛋白,上样的时候加了DTT,结合情况有所改善,但是洗脱下来就比较杂。不过当时我们的蛋白在构建时在GST和目的蛋白之间加了蛋白酶切位点,洗脱下来后切掉tag,再过一次GST亲和柱,蛋白就纯了。
另外,你也可以考虑一下再过一个别的柱子,比如说superdex200, 或者离子柱。我印象里,70kd和30kd的蛋白是可以通过superdex200分开的,你的能不能分开要试了采能知道。