m*********k
发帖数: 10521 | 1 本次统计截止时间为：2012-09-28 02:01:00 （美东时间）
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v*****t
发帖数: 80 | 2 Successfully sent baozi to the following accounts:
['BenArfa', 'thunder8256', 'xybsh', 'HEtorpedo', 'myle', 'xknf', 'playmore',
'happyday365', 'saris', 'aya1', 'sippnet1', 'walkerby', 'ajd', 'godspeedj',
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t****x
发帖数: 1429 | 3 ☆─────────────────────────────────────☆
sks (萨克斯) 于 (Thu Jul 10 14:05:24 2014, 美东) 提到:
NSC. 485 RD 3/10.
7/9， 昨天，终于收到卡了。
这一煎熬过程终于过完了，不用再老是刷屏了。
可是，越来越不确定要不要留在这里～～
☆─────────────────────────────────────☆
robin95 (robin95) 于 (Thu Jul 10 14:05:56 2014, 美东) 提到:
恭喜
☆─────────────────────────────────────☆
arthury (指导实践要科学) 于 (Thu Jul 10 14:06:27 2014, 美东) 提到:
恭喜楼主，
对于最后一个问题，有绿卡也好，可以自由的回国考察机会
☆─────────────────────────────────────☆
xchg99 (xch) 于 (Thu Jul 10 14:07:24 2014, 美东) 提到:
恭... 阅读全帖 |
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r****o
发帖数: 105 | 4 Flag tag 最早来自于T7噬菌体十号基因表达产物11个氨基酸的前导序列。
现在公认的序列是：DYKDDDDK
这个标签的好处是一来比较小，二来比较亲水，所以一般不影响所标记蛋白
的活性。至於是在N还是C端，要看你蛋白的实际情况。比如有些蛋白N端或者C端要
经过蛋白酶切除，这种情况下FLAG显然不能加到要被切除的一端去。当然你也可以
两端都试试，加在哪端活性好，就用那端好了。
如果是N-terminus Flag，ATG应该加在FLAG前面，之前还应该有Kozak sequence,
以便翻译正确。不用隔太多氨基酸，用一两个glycine之类旋转比较灵活的氨基酸
隔开就行了。
如果你想省事，可以找商业化的有FLAG载体，比如Stratagene的
pCMV-Tag系列,既有N端FLAG的载体，又有C端FLAG的载体。又如SIGMA公司的pFLAG-CMV
系列，也是既有N端又有C端FLAG载体。SIGMA公司还提供一种特别的p3XFLAG-CMV系列，
加有三个串联的FLAG。如果你的蛋白做western检测起来信号很弱，加三个或者更多
FL |
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b*********8
发帖数: 44 | 6 我用的FLAG 序列是：DYKDDDDK.难道 就是4个D的版本？我太惨了吧！
我也打算用M2-HRP抗体，是不是多加点抗体会好点，比如1:100稀释？再不行就要用
bead纯化了吧？ |
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h**********r
发帖数: 671 | 10 必须是pESC系列。
当时我还问大家要不要加kozak序列，也没说出个所以然来。后来我终于等到了客服的
答复。下面就是：
The GAL1 and GAL10 yeast promoters from the pESC-Leu vector have the FLAG or
the MYC epitopes in the two MCS of the vector. Both include ATG codons at
the 5’ end of the FLAG and the MYC epitopes. If you clone into the Bgl II
or the Xho I site in frame with the ATG of the epitope tag, you will have
the start codon, kozak sequence and should get good expression of your N-
tagged protein. If you need the epitope tag at the C-terminal, ... 阅读全帖 |
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m******5
发帖数: 1383 | 11 大家有没有遇到过N-terminal tag Flag Tag表达高，但用M2抗体识别不出来，换一个
公司的DYKDDDDK抗体就能识别出来的情况？
Very uncomfortable ！！ |
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f********n
发帖数: 20 | 12 I was wondering if there is FLAG sequence (DYKDDDDK) in any nature protein.
Any body knows? |
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M******g
发帖数: 152 | 13 you can assemble a sequence like this: met-flag tag (DYKDDDDK)- your peptide
. express it in a host like e. coli. Purify the flag-your peptide. you
then use enterokinase to cut off flag tag and leave your peptide with no
scar and no met, since enterokinase recognize DDDDK, and cut after the last
K (you can find more information at NEB: https://www.neb.com/products/p8070-
enterokinase-light-chain).
hope this helps. good luck. |
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发帖数: 1 | 15 flag tag 不是DYKDDDDK吗， 那应该只有1021Da。
120kDa怎么来的？ |
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m*********k
发帖数: 10521 | 16 "[Immigration]
yingying0823 Apr 12 ● [bssd] UPDATE 485求祝福，NSC RD 2/14，今天等来了RFE
祝愿早绿"
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m*********k
发帖数: 10521 | 17 " 【Immigration】
michaelxiaga Jun 27 . ● 我也来求一个485的祝福"
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m*********k
发帖数: 10521 | 18 " 【Immigration】
michaelxiaga Jul 18. ● 散尽家财发包子"
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m*********k
发帖数: 10521 | 19 "[Immigration]xueyamin Oct 25. ● TSC 刚刚收到短信EAD批了（50天），倾家荡产发包子"
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