j*****a
发帖数: 92 | 1 拿有我insert DNA的recombinant plasmid(pRSet A)去测序，１００ ％ 符合。（我的
insert有1.０ kb），transform into BL21(DE3)pLysS Competent Cells 用His柱子纯
化蛋白. SDS-PAGE显示分子量20 kDa, 应该是35 kDa. 谢谢高手们回答。 |
g********4
发帖数: 4959 | 2 There are lots of explanation for MW alteration, and you cannot verify any
explanation without sequencing your purified protein (actually do N-termianl
or C-terminal determination). Some proteins have apparent MW difference on
SDS-gel to calculated MW. Some proteins have beed degradated in cell or
during purification. Some proteins may be made from a inner initial codon (
ATG) if a SD sequence is appripriately placed.You can check the last
possibility by looking at your sequence (but even no SD |
C*********m
发帖数: 213 | 3 SDS-PAGE不准，提出来之后打质谱
【在 j*****a 的大作中提到】
: 拿有我insert DNA的recombinant plasmid(pRSet A)去测序，１００ ％ 符合。（我的
: insert有1.０ kb），transform into BL21(DE3)pLysS Competent Cells 用His柱子纯
: 化蛋白. SDS-PAGE显示分子量20 kDa, 应该是35 kDa. 谢谢高手们回答。
|
s********n
发帖数: 2939 | 4 一般而言SDS-PAGE不会差别这么大的，如果是N-His tag可能是翻译提前终止或降解，
如果是C-His tag可能是降解。 |
